Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1,25-dihydroxy vitamin D₃(D₃) and tri-iodothyronine (T₃) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D₃and T₃treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C₆ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T₃and not with D₃. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D₃and T₃in the osteoblastic MC3T3-E1 and in nonosteogenic cells.