During trophoblast differentiation, cytotrophoblast cells aggregate, fuse and differentiate into syncytiotrophoblast cells that express human placental lactogen(hPL). In a previous study, our laboratory demonstrated that vitamin D3 stimulates hPL gene expression by activation of a VDR element on the hPL promoter. In the present study, we have examined the ontogeny of the VDR during trophoblast differentiation. Cytotrophoblast cells from term human placenta were isolated by enzymatic disaggregation and Percoll gradient fractionation and maintained for 10 days in RPMI 1640 medium containing 10% second trimester pregnancy serum. VDR levels were determined by competitive RT-PCR using a MIMIC constructed with a neutral DNA v-erb B fragment. By day six, 90% of the cells had differentiated into syncytiotrophoblast cells that expressed relatively large amounts of hPL. VDR levels on days 4, 8 and 10 were 5.0, 17.5 and 25.0 fold greater than at the beginning of the culture period, and the pattern of VDR expression paralleled that for hPL. Since VDR and hPL expression increase in parallel during trophoblast differentiation, the study suggests that VDR may be essential to hPL gene expression during trophoblast differentiation. Supported by NIH grant HD-07447.