We rigorously evaluated gender differences in the measurement validity of salivary testosterone. Matched serum, saliva, and finger stick blood spot specimens were collected from 40 (20 males) young adults (aged 18-27 years). Saliva was assayed for testosterone by two independent (isotopic and non-isotopic) immunoassay methods. Serum was assayed by commercially available immunoassay kits for free and total testosterone. An immunoassay was developed for the measurement of testosterone in dried blood spots and is presented in detail so as to be reproducible from this report. Regardless of assay method, salivary testosterone levels are modestly correlated with serum levels for males but not necessarily for females. Blood spot assay results were highly correlated with serum total and free testosterone for both males and females. Substitution of saliva assay results for serum values substantially underestimates known testosterone-behavior associations, and this effect is much more pronounced for females than for males. The findings have important implications for the use and potential misuse of noninvasive measures of testosterone, and with respect to statistical power, the probability of observing significant testosterone-behavior relationships.