We have previously shown that human progesterone receptors (PR) are expressed in human osteosarcoma cells and in primary human osteoblast cultures. The aim of this study was to examine PRa and PRb isoform expression in human osteosarcoma cells. In addition, the effect of beta-estradiol on PR promoter activity in three human osteosarcoma cell lines was analyzed. Rapid amplification of 5'cDNA ends (5'RACE) were used to detect PR mRNA transcripts coding for both PR isoforms in HOS-TE85, an early progenitor human osteosarcoma cell line. Analogous 5'RACE products were detected in the PR-positive breast-cancer cell line MCF-7. Southern blot analysis confirmed that the amplified products were PR specific. It was shown that the larger of two RACE products coded specifically for B isoform mRNA and that of the smaller product corresponded to a PRa specific transcript. No RACE products were detected in the PR-negative HeLa cell line. To determine if both PR promoters were active in osteoblasts, chimeric recombinants bearing the PRa (+464, +1105) and PRb (-711, +31) promoter regions subcloned into minimal pBLCAT vectors, were transiently expressed in three human osteosarcoma cell lines-HOS-TE85, MG-63, and SAOS-2. It was shown that beta-estradiol induced both PRa and PRb promoter activity in all of the osteosarcoma cell lines examined. The finding that PRa and PRb mRNA transcripts are expressed in human osteoblasts, and that promoters for both isoforms are estrogen responsive provides further evidence that bone-forming cells are physiologically influenced by progesterone.