A sex pheromone, protoplast-release-inducing protein (PR-IP), of the Closterium peracerosum-strigosum-littorale complex is known to induce the release of protoplasts from mating-type minus (Mt-) cells during sexual reproduction and to have two subunit polypeptides of 19 and 42 kDa. Here, we describe the regulation mechanism for the release of the PR-IP. The sex pheromone was fractionated to yield subunits of 19 and 42 kDa, respectively, and each subunit was treated with V8 protease and with CNBr. By reference to the partial amino-acid sequences of the digested polypeptides, oligo nucleotides were synthesized and used as primers for the combined reverse transcription-polymerase chain reaction. Amplified fragments of DNA, of 130 bp in the case of the 19-kDa subunit and of 330 bp in the case of the 42-kDa subunit, were obtained, sequenced, and used as probes to identify the respective transcripts. From the results of Northern hybridization, the sizes of transcripts were estimated to be 1.2 kb for the 19-kDa subunit and 1.4 kb for the 42-kDa subunit. These transcripts appeared transiently only when mating-type plus (mt +) cells were treated with another sex pheromone (PR-IP Inducer) for more than 4 h in the light. By immunoblotting with anti-42-kDa-subunit antiserum, it was shown that PR-IP accumulated gradually in the medium but not in the mt + cells after treatment with PR-IP Inducer in the light. We suggest that PR-IP is synthesized de novo and secreted from mt + cells only after the perception of PR-IP Inducer that has been released from mt-cells in the light during the sexual reproduction of Closterium. An analysis by genomic Southern hybridization revealed * Recipient of a Fellowship for Japanese Junior Scientists from the Japan Society for the Promotion of Science