In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e. as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids. The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine: delta 24-sterol-C-methyl-transferase (SMT), a rate-limiting enzyme for phytosterol biosynthesis. A cDNA clone (SMT1) encoding soybean SMT was isolated from an etiolated hypocotyl cDNA library by immunoscreening using an anti-(plasma membrane) serum. The deduced amino acid sequence of the SMT1 cDNA contained three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases. The overall structure of the polypeptide encoded by the SMT1 cDNA is most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene. The polypeptide encoded by the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity. The growing soybean vegetative tissues had higher levels of SMT transcript than mature vegetative tissues. Young pods and immature seeds had very low levels of the SMT transcript. The SMT transcript was highly expressed in flowers. The expression of SMT transcript was suppressed in soybean cell suspension cultures treated with yeast elicitor. The transcriptional regulation of SMT in phytosterol biosynthesis is discussed.