We previously showed that 1α,25-dihydroxyvitamin D3, calcitriol, enhanced phorbol ester 12-O-tetradecanoylphorbol-13- acetate (TPA) induced tumorigenic transformation of mouse epidermal JB6 Cl41.5a cells. To determine if calcitriol regulates this enhancement through a nuclear vitamin D receptor (VDR)-dependent or -independent pathway, we used vitamin D analogs which induce biological responses by either of these mechanisms. In JB6 Cl41.5a cells, 1α,25-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (BT), which like calcitriol binds to VDR and regulates transcription, inhibited cell growth, stimulated expression of nonphosphorylated osteopontin (OPN), and enhanced TPA-induced anchorage-independent growth (AIG, an in vitro assay which highly correlates with tumorigenicity of these cells). 25-Hydroxy-16-ene-23-yne-vitamin D3 (AT), which stimulates calcium influx but has low affinity for VDR, had moderate effects on cell growth and expression of OPN. However, it enhanced TPA-induced tumorigenic transformation, though to a lesser extent than BT, thus suggesting that a VDR-independent mechanism is involved. Since 1α-dihydroxylase activity was detected in JB6 cells, AT could be converted into 1α,25-dihydroxy- 16-ene-23-yne-vitamin D3 (V), an analog which binds with high affinity to VDR, and could subsequently enhance TPA-induced AIG. To verify whether the VDR-independent pathway is involved in calcitriol enhancement of tumorigenic transformation, two additional VDR-independent analogs, 1α,25-dihydroxy-lumisterol3 (JN) and 24R,25-dihydroxyvitamin D3 (AS), were tested. The analog JN, which stimulates calcium transport and cannot be further hydroxylated at 1-carbon position, increased TPA-induced AIG, while AS, which inhibits calcium influx, did not. These studies suggest that a VDR-independent pathway, perhaps stimulation of calcium influx, and a VDR-dependent mechanism, which directly affects transcription, are involved in calcitriol's enhancement of TPA- induced tumorigenic transformation in JB6 Cl41.5a cells.