A variant of the chronic myelogenous leukemia cell line, RWLeu-4, that is resistant to the antiproliferative effects of vitamin D3 was established. Although RWLeu-4 proliferation is inhibited by 1 nmol/L vitamin D3, the resistant cells (JMRD3) continue to proliferate in the presence of 100 nmol/L vitamin D3. Both cells express similar patterns of differentiation-specific antigens after treatment with vitamin D3, and both express the retinoblastoma gene product (p110Rb). Vitamin D3 treatment of the sensitive RWLeu-4 cells decreased the level of the p110Rb protein, as well as its phosphorylation. In contrast, vitamin D3 treatment of JMRD3 had no effect on p110Rb expression or phosphorylation. Both RWLeu-4 and JMRD3 express similar vitamin D3 receptors and vitamin D3-inducible enzyme activities. Differences were detected in the DNA binding characteristics of the vitamin D3 receptors as determined by electrophoretic mobility shift studies. However, sequence analysis of the DNA-binding domain and immunoblot analysis showed no differences in the receptors. We conclude that some process subsequent to vitamin D3 receptor activation is altered in JMRD3 that partially separates vitamin D3-induced inhibition of proliferation from the induction of differentiation.