Pyridoxal phosphate (PLP), a bio-active form of pyridoxine, (10-1000 µM) dose-dependently inhibited cell proliferation in rat pituitary MMQ and GH3 cells, and in mouse AtT-20 cells. After 4 days, MMQ cell numbers were reduced by up to 81%, GH3 cell numbers were reduced by up to 64% (P < 0.05) and AtT-20 cell numbers were reduced by up to 90% respectively. Cell proliferation rates recovered and dose-dependently reverted to control levels after PLP withdrawal. After 4 days, PLP (400 and 1000 µM) decreased H-3-thymidine incorporation by up to 71% (P < 0.05). PLP (400-1000 µM) reduced GH3 cell GH and PRL secretion, and AtT-20 cell ACTH secretion (adjusted for cell number) by 70% after 2 days. 100 µM PLP also inhibited PRL secretion (65%, P < 0.05) in primary rat pituitary cells treated for 2 days. PLP decreased the percentage of AtT-20 and GH3 cells in S phase, and increased those in GO-G1 phase. Furthermore, PLP induced AtT-20 and GH3 cell apoptosis (28 vs. 6, P < 0.05; 26 vs. 3, P < 0.05, respectively), and dose-dependently reduced content of the anti-apoptosis gene, Bcl-2. These results indicate that pharmacologic doses of PLP inhibit pituitary cell proliferation and hormone secretion, in part mediated through PLP-induced cell-cycle arrest and apoptosis. Pyridoxine may therefore be appropriate for testing as a relatively safe drug for adjuvant treatment of hormone-secreting pituitary adenomas.