Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin superfamily, with recognizable homologues in all fish taxa examined to date. Although sequences from most fish share reasonably high sequence identity, several more highly divergent SLs have been reported. Goldfish SL and a second SL protein found in rainbow trout (rtSLP) are remarkably different from each other and also dissimilar to other SLs. It has been unclear whether rtSLP is a recent paralogue restricted to rainbow trout, or reflects a more ancient duplication of the SL gene, and whether it is related to the goldfish sequence. Here we report the cloning of two different zebrafish SL cDNAs, which share only 57·5% nucleotide and 47·7% deduced amino acid identities. One copy, designated zebrafish SL (zfSL ), displays a typical range of sequence similarity to most other SLs. The other copy, zebrafish SL (zfSL ), shows low identity to most other SLs; surprisingly, it is most similar to the divergent SL sequence from goldfish. The mRNAs of zfSL and zfSL were expressed specifically in two distinct regions of the pars intermedia in zebrafish. Cells expressing zfSL are located at the posterior pars intermedia, bordering the neurohypophysis, whereas zfSL is expressed in the anterior part of the pars intermedia, bordering the pars distalis. Phylogenetic analyses indicate that zfSL , goldfish SL and rtSLP all belong to the SL hormone family; however, along with the genes from eel and catfish, these divergent sequences form a group that is clearly distinct from all other SLs. These results suggest the presence of two distinct SL families, SL and SL , which may trace back to a teleost genome duplication prior to divergence of the cyprinids and salmonids.