In male Japanese eels, eel spermatogenesis-related substance (eSRS) 3 and 4, having high sequence-similarities to zona pellucida protein (ZP) 2 and ZP3, respectively, are down-regulated by gonadotropin stimulation, with their transcripts disappearing upon the initiation of spermatogenesis. Using Northern blot analysis, we investigated the expression of eSRS3 and 4 mRNA in the developing ovary and the liver of SPH (salmon pituitary homogenate)-injected female eels. Both transcripts were detected in the ovary, but not in the liver. When the eel ovary was subjected to in situ hybridization using eSRS3 and 4 cRNA probes, the cytoplasm of previtellogenic oocytes showed a strong signal in comparison with the weak signal in vitellogenic oocytes. Furthermore, stronger signals were observed in the chromatin-nucleolus and the perinucleolus stages than in the oil-droplet stage. Subsequently, we synthesized peptides that were deduced from eSRS3 and 4 cDNAs and generated specific antibodies against them. Staining of the cytoplasm of oocytes in the previtellogenic stage and of egg envelopes in the vitellogenic stage occurred when these antibodies were used in an immunohistochemical analysis. These expression patterns in the ovary suggest that eSRS3 and 4 are components of the eel egg envelope.