A cell line, RTP-2, has been developed from a normal-appearing pituitary of an adult rainbow trout. The cells grow in L-15 basal medium, supplemented with 2.5% to 10% fetal bovine serum, and have been passaged approximately 50 times over a 2-year period. At low density the cells have a stellate shape, whereas at confluency islands of polygonal cells appear among a tangle of bipolar cells. At the ultrastructural level, most cells contain numerous lysosomes, autophagic vacuoles, and intermediate filaments, but no obvious secretory granules. Reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotides specific for growth hormone (GH), prolactin (PRL), and somatolactin (SL) as amplification primers and Southern blot hybridization of the PCR products with probes specific for GH, PRL, and SL demonstrate that this cell line expresses GH, PRL, and SL. Digestion of the GH product of RT-PCR with restriction endonuclease SalI or KpnI confirms that both rainbow trout growth hormone genes are expressed in this cell line.