Agouti protein (AP) expression in the wild-type agouti mouse (AwJ/AwJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (Ay/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of Ay/a, AwJ/AwJ, and a/a (black) mice, and 2) adipose and spleen tissues of Ay/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. Ay/a hair follicles and serum showed highest cysteine and lowest GSH levels. AwJ/AwJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed Ay/a > a/a (P < 0.01). An Ay-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.