The National Union of Teachers is threatening legal action against an Essex school it claims routinely uses teaching assistants to take classes on their own.
The case against St Thomas More, Southend, would be seen as a key test for schools that want to press ahead with implementing the workload agreement.
But it would be unlikely to have a long-term impact on the agreement as ministers are preparing new regulations which specifically allow assistants to take classes.
St Thomas More is one of a number being targeted by the NUT, the only union to refuse to sign the workload agreement.
Frank Keenan, head of the Catholic boys' secondary school, said he could not comment on the issues because of an employment tribunal case involving a former supply teacher.
But he did say that the school is "vigorously fighting these suggestions".
The union is also writing to Swavesey village college, Cambridgeshire, after the school sent an email to parents inviting applications for a pound;56 per day classroom supervisor post. Duties include covering for "short-term illnesses, professional development activities and other absences", the message said. "The post would be ideally suited to individuals who have experience of working with young people, though full training and support will be provided."
Martin Bacon, headteacher, said that the school was piloting the new approach because it could not guarantee the quality of supply teachers and wanted to improve the "continuity of learning" for pupils.
Supervisors would only cover for short-term absences during a pilot period covering the second half of the summer term, he said.
Norman first school in Norwich will also receive a letter from the NUT concerning claims, denied by the headteacher, that an unqualified assistant is starting a second term in charge of a reception class.
Hilary Bucky, regional officer of the NUT, said that the union is considering legal action following St Thomas More's employment of two teaching assistants to replace a supply teacher before Christmas.
A spokeswoman for the union said: "The use of classroom assistants in place of qualified teachers will jeopardise children's education."