Campaigners against a new motorway south of Newport have held a rally at the Senedd, claiming the plans would harm wildlife and increase pollution.
The Campaign Against Levels Motorway (Calm) want proposals for the 15 mile (24km) toll road dropped.
Julian Branscombe, the group's chair, said the road would be "a major step backwards in terms of protecting sites for wildlife".
The assembly government said the business case was still being prepared.
The aim of the project would be to reduce congestion between junctions 23A and 29 and relieve traffic build-up on the stretch of the M4 through Newport, which includes the Brynglas tunnels.
The two-lane motorway through the city does not meet current motorway standards and traffic already exceeds capacity at peak times.
Following two fatal crashes in the area, including a head-on collision where five people died, Transport Minister, Ieuan Wyn Jones, said last year the new motorway could open by 2013.
The effect of this levels motorway on Wales' most important area for grazing marsh wildlife would be catastrophic
Julian Branscombe, chair of Calm
If built, the road would stretch between Magor in Monmouthshire and Castleton near Newport and run across a number of sites of special scientific interest (SSSIs).
But Mr Branscombe said: "The Welsh Assembly Government has already highlighted fragmentation of SSSIs as a major threat to biodiversity in Wales - the effect of this levels motorway on Wales' most important area for grazing marsh wildlife would be catastrophic."
Haf Elgar, of Friends of the Earth Cymru, said the group was also convinced the relief road was "unnecessary, and will soon be seen to be unaffordable".
"This six-lane toll road would do little to improve safety or tackle congestion on the M4, but it would take our concentration away from the sustainable transport solutions we should be pursuing," she added.
"Rather than vastly expensive road building schemes, we need to be moving more of our transport spend into public transport and traffic reduction. Any sensible consideration will see this motorway proposal rejected immediately."
But Mike Farmer, of the Road Haulage Association, said: "The congestion on the current motorway is probably causing more pollution than there would be on a relief road.
"The economy of south Wales is wholly dependant on having good east-west road access and we believe a relief road is needed."
A spokesperson for the assembly government said: "The business case for this scheme is currently being prepared. The project planning also includes a series of environmental impact surveys, required by law.
"If the business case is approved by the minister, it is expected that a public local inquiry will be held, which will provide an opportunity for an independent review.
"The recommendations from this inquiry will enable the minister to make an informed decision."
Member organisations of Calm include the Campaign for the Protection of Rural Wales, Gwent Wildlife Trust, Friends of the Earth Cymru, RSPB Cymru and WWF Cymru, and local community councils.
The rally took place on the steps of the Senedd on Tuesday attracting around 180 protesters.
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