We reported previously the identification of three homeobox-containing genes, prox1, prox2 and prox3, in sponges [Seimiya, M., Ishiguro, H., Miura.,K., Watanabe, Y. & Kurosawa, Y. (1994) Eur. J. Biochem. 221, 219-225]. The transcripts of prox1 and prox2 were identified in cells at all stages of differentiation. In the present study, we have identified two POU-class homeobox genes, designated spou-1 and spou-2, in a freshwater sponge (Ephydatia fluviatilis). These genes each encode a POU-specific domain and a POU-type homeodomain. The amino acid sequences of the POU-specific domain and the POU-type homeodomain encoded by the spou-1 gene were 76% and 67% similar to those of the human Pit-1 gene, respectively. The amino acid sequence of the POU-specific domain encoded by the spou-2 gene was also most similar to that encoded by the human Pit-1 gene among all the POU-class homeobox genes that have been sequenced to date. In contrast to the results for prox1 and prox2, transcripts of the spou-1 and spou-2 genes were identified in cells only at specific stages during the differentiation of the sponge.