Five single-cell clone lines (mRTP1B, mRTP1E, mRTP1F, mRTP1K, and mRTP2A) have been developed from adult rainbow trout pituitary glands. These cell lines have been maintained in a CO 2 -independent medium supplemented with 10% fetal bovine serum (FBS) for more than 150 passages. At about 150 passages, the doubling time of each single-cell clone in a CO 2 -independent medium supplemented with 10% FBS at 20 8C was 3 . 6G0 . 7, 2 . 8G0 . 7, 3 . 2G0 . 8, 5 . 5G0 . 6, and 6 . 6G0 . 6 days respectively. Each single-cell clone contains 60G2 chromosomes, which is within the range of the 2N chromosome numbers reported for rainbow trout. Reverse transcription-PCR analysis revealed that in addition to expressing gh, prolactin (prl ), and estradiol (E 2 ) receptor a (e2ra or esr1) genes, each single-cell clone line also expressed other pituitary-specific genes such as tsh, gonadotropin 1 (gth-1 or fshb), gonadotropin 2 (gth-2 or lhb), somatolactin (sl or smtl ), proopiomelanocortin-B (pomcb), and corticosteroid receptor (cr or nr3c1). Immunocytochemical analysis showed that all the five single-cell clones produced both Gh and Prl. Furthermore, the expression of gh and prl genes in the single-cell clone lines is responsive to induction by E 2 , dexamethasone, and o,p 0 -dichlorodiphenyltrichloroethane. All together, these results confirm that each of the singlecell clones was derived from rainbow trout pituitary glands. These single-cell clone lines not only can be used to study factors that regulate the expression of pituitary hormone genes, but can also be developed as a rapid screening system for identifying environmental endocrine disruptors.