Several theories have been advanced to explain hair graying and whilst predisposition to this trait is emerging as largely genetic, hypotheses for the mechanism include reactive oxygen species (ROS)-associated damage in hair follicle melanocytes (HFM) coupled with an impaired anti-oxidant status and a failure of melanocyte stem cell renewal. We have now examined age-associated differences between fully-matched HFM and epidermal melanocyte (EM) cultures focusing on proliferation, expression of melanogenic and anti-oxidant enzymes and impact of oxidant stress on expression and activity of the anti-oxidant enzyme catalase. Proliferation of HFM was higher than EM but HFM were more affected by aging. Melanogenic enzyme expression decreased with age and for tyrosinase this was greater in HFM than EM. Expression and activity of catalase was similar in younger HFM and EM, but was markedly down-regulated with age in HFM. No age-related change in catalase expression/activity was observed in matched skin dermal fibroblasts. Melanocyte survival, catalase expression and activity, and the expression of p38 stress kinase were examined after exogenous and endogenous exposure of HFM to oxidant stress (hydrogen peroxide, H2O2). Catalase expression and activity was up-regulated by H2O2 in young donor, but not older donor HFM, and we found higher levels of p38 stress kinase expression in the latter compared to young donor HFM. The addition of anti-oxidants (Vitamin E and Quercetin) to H2O2-treated HFM from older donors resulted in increased cell survival and partially restored catalase expression and activity. Finally, using three differently pigmented melanoma cell lines, we found that expression and activity of catalase correlated directly with (and was dependent on) melanogenic activity. In summary, we propose that the reduction in catalase in HFM with age supports the hypothesis that it is the age-related disruption of anti-oxidant defence in HFM that contributes to the development of hair graying.