Aromatase cytochrome P450 (CYP19), which is necessary for the conversion of androgens to estrogens, plays an important role in the sexual differentiation of the brain. To investigate whether differences in the gene encoding the aromatase enzyme influence sexual orientation in men, we conducted linkage, association, and expression analyses in a large sample of homosexual brothers using microsatellite markers in and around CYP19. No linkage was detected, and a gene-specific relative risk of 1.5-fold could be excluded at a lod score of -2. Results of the TDT demonstrated no preferential transmission of any of the CYP19 alleles in this sample. Expression of aromatase mRNA by microarray analysis was not significantly different between heterosexual and homosexual men. These results suggest that variation in the gene for this subunit of the aromatase enzyme complex is not likely to be a major factor in the development of individual differences in male sexual orientation.