S-Adenosylhomocysteine hydrolase (AHCY) is the only mammalian enzyme known to catalyze the hydrolysis of S-adenosylhomocysteine. We have used a genotype-to-phenotype strategy to study this important enzyme by resequencing AHCY in 240 DNA samples from four ethnic groups. Thirty-nine polymorphisms were identified - 28 of which were novel. Functional genomic studies for wild type AHCY and the three variant allozymes identified showed that two variant allozymes had slight, but significant decreases in enzyme activity, but with no significant differences in levels of immunoreactive protein. Luciferase reporter gene assays for common 5'-flanking region haplotypes revealed that one haplotype with a frequency of approximately 2% in Caucasian-American subjects displayed a decreased ability to drive transcription. The variant nucleotide at 5'-flanking region single nucleotide polymorphism (SNP) (-34) in that haplotype altered the DNA-protein binding pattern during electrophoresis mobility shift assay. Finally, an AHCY genotype-phenotype association study for expression in lymphoblastoid cells identified four SNPs that were associated with decreased expression. For the IVS6 (intervening sequence 6, i.e., intron 6) G56 > C SNP among those four, electrophoresis mobility shift assay showed that a C > G nucleotide change resulted in an additional shifted band. These results represent a step toward understanding the functional consequences of common genetic variation in AHCY for the regulation of neurotransmitter, drug and macromolecule methylation.