The significance of conserved cysteines at positions 288, 337, and 369 in the hormone binding domain of the human vitamin D receptor was evaluated by individual site-directed mutagenesis to glycine. Neither nuclear localization nor heterodimerization with retinoid X receptors in binding to the vitamin D-responsive element was appreciably affected by altering these cysteines, but vitamin D hormone (1,25-(OH)2D3) activated transcription was compromised significantly in the C288G and C337G mutants. Only the C288G mutant displayed depressed (3-fold) 1,25-(OH)2D3 ligand binding affinity at 4 degrees C, in vitro, although at elevated temperatures (23-37 degrees C), ligand binding was attenuated severely in C288G, moderately in C337G and very mildly in C369G. The degree of impairment of ligand binding at physiologic temperatures correlated with the requirement for increased concentrations of 1,25-(OH)2D3 ligand to maximally stimulate transcriptional activity in co-transfected COS-7 cells. Thus cysteine 288 and, to a lesser extent, cysteine 337 are important for high affinity hormone binding to the vitamin D receptor, which ultimately leads to ligand-dependent transcriptional activation.