During fertilization in Chlamydomonas, flagellar adhesion between mt+ and mt- gametes induces a cAMP-dependent signal transduction pathway that prepares the gametes for cell fusion and zygote formation. Previously, our laboratory identified a homeodomain protein (GSP1) whose expression was restricted to the cell bodies of mt+ gametes and whose transcript level was upregulated during flagellar adhesion. In this report, we describe a new form of GSP1 that appears early during gamete interactions. Immunoblot analysis showed that in addition to the 120-kDa form of GSP1 normally present in mt+ gametes, a 122-kDa form was detected when the cells were mixed with mt- gametes. The more slowly migrating form of GSP1 was detectable within minutes after gametes were mixed together, and its appearance did not require new protein synthesis. Thus, the 122-kDa form represents a post-translational modification of the pre-existing 120-kDa form of GSP1. Moreover, conversion to the 122-kDa form did not require cell fusion. Although the 120-kDa form was expressed 10 h after vegetative cells were transferred to gametic induction medium, the 122-kDa form was detected only after mt+ gametes were induced to undergo the sexual signaling that accompanies fertilization. Incubation of mt+ gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase inhibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 immunoprecipitated from signaling mt+ gametes with alkaline phosphatase showed that the conversion was due to phosphorylation. The results indicate that flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of the homeodomain protein GSP1 early during fertilization in Chlamydomonas.