5,5-diphenylhydantoin (Phenytoin, PHT), a widely used anticonvulsant, is also a vitamin K antagonist and disrupts bone metabolism, leading to osteomalacia. The vitamin K-dependently synthesized protein, osteocalcin, has been implicated as a key regulatory protein in bone resorption. The purpose of the present study was to determine whether PHT had an effect on osteocalcin secretion. Cells were grown to confluence in Ham's F-12 nutrient mixture, and treated with 1,25 (OH)2 vitamin D3 (2.6 microM to 2.6 pM) or PHT (5-100 micrograms/mL) for either 24 or 48 h of pretreatment. The media were then discarded, replaced with fresh media and test reagents, and quantitated for osteocalcin by radioimmunoassay at 0, 4, and 8 h secretion time points. Results were statistically analyzed by the Student's two-tailed t test. Controls showed a nearly linear secretion rate of osteocalcin, reaching 8-9 ng/10(6) cells by 8 h. Vitamin D3 (2.6 nM) maximally stimulated secretion nearly two-fold after 24 or 48 h of pretreatment in comparison to controls. PHT alone (25-100 micrograms/mL) exerted an inhibitory effect, which appeared dose-dependent and was most evident at 4 and 8 h. PHT (50 micrograms/mL) had a significant effect, in the presence of a range of vitamin D3 concentrations (2.6 microM to 2.6 pM), after 48 h of pretreatment. A maximal PHT dose of 100 micrograms/mL had no effect on either the viability or the numbers of cultured cells. These data indicate that PHT affects osteocalcin secretion from osteoblastic rat osteosarcoma (ROS 17/2.8) cells.