To examine effects on cell proliferation, osteopontin (OPN) protein and mRNA expression in two different bone cell lines, we treated cells with lOnM calcitriol (C) or its analogs [calcipotriol (BT), 25-OH-16-ene-23-yne-D3 (AT), 25(OH)D3 (BO), and 24,25(OH)2D3 (AS)] for 24 hours. In ROS 17/2.8 cells there were no significant differences in cell proliferation, but calcitriol and all analogs had an inhibitory effect in MC3T3 cells. TCA precipitation after metabolic labeling with 32PO4 showed that C. BT and AT increased the level of total phosphorylated protein in both cell lines. Immunoadsorption assays to assess OPN protein expression showed that compared to control cells, C and BT, but not AT, BO, AS stimulated the expression of OPN protein Northern blots demonstrated that only C and BT increased OPN mRNA expression These findings suggest that some analogs of calcitriol can stimulate genomic and nongenomic pathways in osteoblast-like cells but that effects vary with cell line. Supported by NIH DE067.W.