The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the physiologically active form of vitamin D3 that inhibits proliferation and induces differentiation of a variety of malignant cells. We evaluated a newly synthesized vitamin D3 analogue [1,25(OH)2-16-ene-5,6-trans-D3 (Ro 25-4020)] that has a novel 5,6-trans motif. Dose-response studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 had 10-100-fold greater antiproliferative activities than 1,25(OH)2D3 when measuring clonal growth of breast (MCF-7) and prostate (LNCaP) cancer cell lines as well as a myeloid leukemia cell line (HL-60). Because the chief toxicity of vitamin D3 is hypercalcemia, we examined the calcemic activity of 1,25(OH)2-16-ene-5,6-trans-D3 in mice. Remarkably, 1,25(OH)2-16-ene-5,6-trans-D3 was at least 40-fold less calcemic as compared with 1,25(OH)2D3 and 1,25(OH)2-16-ene-D3 (Ro 24-2637). To explore the mechanism by which the 1,25(OH)2-16-ene-5,6-trans-D3 analogue mediated its antiproliferative activity, several studies were performed. Pulse-exposure studies showed that a 4-day pulse exposure to 1,25(OH)2-16-ene-5,6-trans-D3 (10(-7) M) in liquid culture was adequate to achieve a 40% inhibition of MCF-7 clonal growth in the absence of the analogue, suggesting that the growth inhibition mediated by 1,25(OH)2-16-ene-5,6-trans-D3 was at least in part irreversible. Cell cycle studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 increased the proportion of MCF-7 cells in the G0-G1 phase and decreased those in the S phase. Furthermore, 1,25(OH)2-16-ene-5,6-trans-D3 induced an elevated expression of the cyclin-dependent kinase inhibitors, p21waf1 and p27kip1. In addition, 1,25(OH)2-16-ene-5,6-trans-D3 almost completely inhibited telomerase activity, as measured by telomeric repeat amplification protocol assay and human telomerase reverse transcriptase mRNA. For each of the growth-related parameters that were examined, the vitamin D3 analogue was more active than 1,25(OH)2D3. In contrast, 1,25(OH)2D3 was more calcemic than 1,25(OH)2-16-ene-5,6-trans-D3. In summary, 1,25(OH)2-16-ene-5,6-trans-D3, having a novel 5,6-trans motif, strongly inhibited clonal proliferation and reduced telomerase activity with low calcemic activity, suggesting further testing in in vivo cancer models. This analogue may gain a therapeutic niche for selected malignancies.