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The roe deer is a seasonally breeding species with a reproductive cycle regulated by endogenous rhythms and photoperiod-sensitivity. Sexually mature bucks show hormonal and testicular activation during the reproductive season, with a peak in the rut period, and following gradual involution. Hair is a good matrix for non-invasive endocrinological analyses that provide long-term information without being influenced by the hormones&rsquo, pulsating release patterns in blood. The aim of the work was to quantify hair concentrations of testosterone and cortisol in wild roe deer bucks hunted during the pre- and post-rut period, using a radioimmunoassay methodology, and to look for differences between the two periods. The secondary objective was the evaluation of possible correlations of such hair concentrations with blood and morphometric parameters of the testes. Both hormones showed statistical differences, with opposing trends, when comparing the two periods: testosterone increased while cortisol decreased. The correlation analysis was in agreement with existing literature regarding metabolism/actions of these hormones and testicular morphometric parameters. This study represents the first report of the use of radioimmunoassay techniques to quantify testosterone and cortisol in roe deer hair, and may provide interesting insights into their reproductive physiology.