The petition relates to Reverend Scott Rennie
A fifth of all Church of Scotland clergy have signed an online petition against the appointment of an openly gay minister, BBC Scotland can reveal.
Concern has been raised within the Kirk that failure to overturn the appointment could provoke a mass walkout by many members.
The petition relates to Reverend Scott Rennie, the minister at Queen's Cross Church in Aberdeen.
The Church's General Assembly will rule on the case later this month.
The online petition states: "We urgently alert all commissioners to the 2009 General Assembly to the extreme gravity of the situation.
"We urge the Assembly to support the position of those who stood to defend Christian orthodoxy in Aberdeen Presbytery, and ensure instead that the Church will apply and assert in practice its clear doctrinal position on all matters of marriage and human sexuality, by refusing to condone homosexual practice in general, and among its leaders in particular."
It has attracted support from more than 230 Kirk ministers, almost 2,000 Church of Scotland members and more than 3,000 other church members around the globe.
Mr Rennie's parish have said they are happy with their new minister and believe that the national Kirk should be a broad church, encompassing a wide range of beliefs and lifestyles.
The Church of Scotland considered the viability of gay relationships two years ago, and decided on a period of reflection.
One of the petition's organisers is the Rev Steven Reid, the minister for Crossford and Kirkfieldbank church in Lanarkshire.
He told BBC Scotland's Newsdrive programme he would remain inside the Kirk whatever the decision, but that he fears a major schism could form.
He said: "The minute you're talking about the division of a church over areas that are deeply held on both sides of the discussion, you realise there's the possibility of that division happening and you can't rule it out."
Mr Rennie's session clerk, Professor Trevor Salmon, said: "If you've got a national church, you have to be broad. We've obeyed the laws of the Church of Scotland, the right of the congregation to call a minister has been there since about 1843.
"The congregation has supported it, the presbytery has supported it, so it's not an idiotic, wacky decision."
Last month, the Kirk's in-house magazine Life and Work called on believers to come to terms with gay relationships.
The Church said the magazine was editorially independent, and its views were not necessarily those of the Kirk.