A number of studies have demonstrated the ability of retinoic acid (RA) to inhibit the growth of estrogen receptor-positive (ER+) human breast cancer cell lines. The precise mechanism of growth inhibition is not known. However, the biological effects of RA in other model systems have been shown to be mediated via the nuclear retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). While several laboratories have examined the expression of RARs in various breast cancer cell lines, no information is available concerning the role of the RXRs and 9-cis-RA, the natural ligand of RXRs, in the response of breast cancer cells to RA. Using a representative panel of breast cancer cell lines, we determined the effect of 9-cis-RA on growth and cell cycle stage distribution, analyzed steady-state mRNA levels of RXR-alpha, -beta, and -gamma, and determined the effect of all-trans-RA and 9-cis-RA on RXR expression. Our results show that: (1) the growth of ER+/RA-sensitive breast cancer cells is inhibited by treatment with 9-cis-RA by blocking entry into S phase; (2) both ER+/RA-sensitive and ER-/RA-resistant breast cancer cell lines express RXR-alpha and RXR-beta mRNAs but not RXR-gamma; however, levels of these transcripts did not correlate with the RA response; and (3) levels of RXR-alpha and RXR-beta mRNA were not significantly altered following treatment with either all-trans-RA or 9-cis-RA. These results suggest that the mechanism responsible for the retinoid sensitivity of breast cancer cells does not involve transcriptional modulation of the RXRs by RA.