Natural gossypol (GP), a polyphenolic pigment in cottonseed, is a racemic mixture of two enantiomers, (+)GP and (-)GP. Our aim was to compare the abilities of (+/-)GP, (+)GP and (-)GP to reduce proliferation of breast cancerous epithelial cells (cEC) and cancerous stromal cells (cSC). Proliferation was measured by 3H-thymidine uptake. Results showed that (+)GP had no effect on both cEC and cSC. In contrast, in both cell types, (+/-)GP and (-)GP significantly inhibited proliferation. (+/-)GP caused reductions of 15, 46 and 82% at 25, 5.0 and 7.5 microM, respectively, in cEC, and reductions of 17, 28, 39 and 56% at 2.0, 3.0, 4.0 and 5.0 microM, respectively, in cSC. (-)GP induced reductions of 33, 89 and 98% at 2.5, 5.0 and 7.5 microM, respectively, in cEC, and reductions of 29, 51, 64 and 72% at 2.0, 3.0, 4.0 and 5.0 microM, respectively, in cSC. By RT-PCR, we found that 3 microM of (+/-)GP and (-)GP decreased cyclin D1 mRNA expression in both cell types (52% and 71%, respectively, in cEC; and 47% and 71%, respectively, in cSC), and increased transforming growth factor beta (TGFbeta) mRNA expression in both cell types (93% and 130%, respectively, in cEC; and 45% and 89%, respectively, in cSC). Interestingly, (-)GP was significantly more potent than (+/-)GP. These results show that (-)GP is the major inhibitory component of (+/-)GP, (-)GP is the more potent inhibitor of cancerous breast cell growth, and the inhibitory activity of (-)GP and (+/-)GP is related to the reduction of the cell cycle regulator, cyclin D1, and the induction of the cell proliferation inhibitor, TGFbeta.