The antiproliferative effect of 1alpha,25(OH)(2)D-3 on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D-3 and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D-3 and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96 h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)2D3 had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48 It by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D-3 was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a C-14-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D-3 on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D3 failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D3 was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D3 regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D3 is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D3 antiproliferative effect in prostate cancer LNCaP cells. (C) 2004 Elsevier Inc. All rights reserved.