Cloning of the gametolysin gene, mmp1, of Chlamydomonas reinhardtii revealed that the gene has six introns in its coding region and that a novel structurally related gene. mmp2, is located just upstream of the mmp1 gene. The mmp2 gene has ten introns and the deduced amino acid sequence has the hallmark motif characteristics of metzincin-type matrix metalloproteases. Southern analysis showed that mmp1 and mmp2 are single copy genes and that a RFLP is found between the mt+ and mt- strains employed. Northern analysis revealed that the two genes are expressed at different stages in the Chlamydomonas sexual cell cycle: steady-state levels of mmp1 mRNA increased during gametogenesis, while those of mmp2 mRNA increased in young zygotes. Transcription of mmp2 was insensitive to cycloheximide and did not occur when a non-fusing mutant, fus mt+, was mated with wild-type mt- gametes, suggesting that mmp2 is a member of the early zygote-specific genes, which are under the regulation of transcription factors pre-existing in gametes and activated by cytoplasmic fusion.