The role of RARα1 and RARγ2 AF‐1 and AF‐2 activation functions and of their phosphorylation was investigated during RA‐induced primitive and parietal differentiation of F9 cells. We found that:(i) primitive endodermal differentiation requires RARγ2, whereas parietal endodermal differentiation requires both RARγ2 and RARα1, and in all cases AF‐1 and AF‐2 must synergize;(ii) primitive endodermal differentiation requires the proline‐directed kinase site of RARγ2‐AF‐1, whereas parietal endodermal differentiation additionally requires that of RARα1‐AF‐1;(iii) the cAMP‐induced parietal endodermal differentiation also requires the protein kinase A site of RARα‐AF‐2, but not that of RARγ; and (iv) the AF‐1‐AF‐2 synergism and AF‐1 phosphorylation site requirements for RA‐responsive gene induction are promoter context‐dependent. Thus, AF‐1 and AF‐2 of distinct RARs exert specific cellular and molecular functions in a cell‐autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation.