Human breast cancer cell lines expressing the estrogen receptor alpha (ERalpha), all-trans-retinoic acid (ATRA) receptor alpha (RARalpha) and cellular retinoic acid binding protein II (CRABPII) genes are sensitive to ATRA-mediated growth inhibition. To study the relationship among ERalpha, RARalpha and CRABPII expression, the protein levels of each member were compared in five breast cancer cell lines (T47D, MCF-7, ZR-75-1, Hs587 T and MDA-MB-231 cells) and two immortalized nontumorigenic breast epithelial cell lines (MTSV1.7 and MCF-10A). ERalpha, RARalpha and CRABPII proteins were detected in T47D, MCF-7 and ZR-75-1 cells but not in other tested cell lines. RARalpha and CRABPII proteins were either reduced or undetectable in T47D/C4:2W and MCF-7/ADR cells with lost expression of ERalpha. Estradiol increased and anti-estrogens (tamoxifen and ICI 164,384) downregulated the expression of both RARalpha and CRABPII proteins in T47D and MCF-7 cells. RARalpha antagonist Ro-41-5253 inhibited CRABPII expression, but not RARalpha expression in estradiol-treated T47D and MCF-7 cells. Suppression of ERalpha by small interfering RNA (siRNA) reduced RARalpha and CRABPII gene expression and siRNA suppression of RARalpha reduced CRABPII expression while having no effect on ERalpha in T47D cells. Transient transfection of either RARalpha or ERalpha expression vectors increased CRABPII expression in MDA-MB-231 cells but only RARalpha, not ERalpha, activated hCRABPII promoter reporter. These results indicate that there is a gene activation pathway in which ERalpha drives RARalpha transcription and RARalpha drives CRABPII transcription in ERalpha-positive human breast cancer cells.