The effects of the immunosuppressive drug cyclosporin A (CsA) were evaluated on ROS 17/2.8 cells in vitro. ROS cells were treated with CsA (0, 0.5, 1.0, 5.0 micrograms/ml) for 3 days with and without bovine parathyroid hormone (bPTH) (1-34) 10 nM. CsA at 0.5, 1.0, 5.0 micrograms/ml without PTH and at 5.0 micrograms/ml in the presence of PTH significantly inhibited proliferation, as determined by a tetrazolium colorimetric assay. In addition, ROS cell number was significantly reduced at 3 and 4 days with CsA (5.0 micrograms/ml) without affecting cell viability. Incorporation of [3H]-thymidine into DNA was significantly reduced by 3.0 and 5.0 micrograms/ml CsA after 12 and 24 hours exposure. Basal and 1,25-dihydroxyvitamin D3-stimulated alkaline phosphatase levels in confluent ROS cells were reduced (P less than 0.05) with CsA (1.0 and 3.0 microgramS/ml). Pretreatment of ROS 17/2.8 cells with CsA did not alter PTH-stimulated cAMP levels or [125I]-PTHrP binding to ROS cells. CsA treatment of ROS 17/2.8 cells induced a spindle-shaped appearance with loss of attachment in confluent cultures. When ROS cells were cultured in CsA-containing media, cellular attachment at 6 and 12 hours was reduced (P less than 0.05) compared with untreated ROS cells. These findings indicate that CsA was capable of inhibiting proliferation, cell number, mitogenesis, alkaline phosphatase levels, and cell attachment of ROS cells without affecting PTH binding or cAMP levels. This direct effect of CsA on osteoblasts may be important in changes of bone remodeling observed in CsA-treated humans and animals.