Seven normal volunteers (six males and one female) with tanning skin types III or IV (Fitzpatrick scale) were given 10 daily subcutaneous injections of a superpotent synthetic analog of alpha-melanocyte stimulating hormone (α-MSH) over two weeks. This agent, [Nle⁴-d-Phe⁷]α-MSH, also called Melanotan-I (MT-I), was administered at a dose of 0.16 mg/kg/day (Monday–Friday), over a two week period. Tanning was measured serially using computerized light reflectance. This regimen induced tanning at 3 of 8 anatomic sites including the face, neck and forearm by comparison of baseline to (1) the end of the daily dosing period, (day 14), and (2) one week later, (day 21). Shave biopsies of the forearm taken at baseline and day 21 were analyzed by high performance liquid chromatography for eumelanin content which was measured as the permanganate oxidation product, pyrrole-2,3,5-tricarboxylic acid or PTCA. Pheomelanin content was measured as the hydroiodic acid digestion product, aminohydroxyphenylalanine (AHP). Eumelanin was also measured in the forehead skin samples of three subjects. The HPLC results show that mean (±SD) baseline eumelanin (PTCA) levels in forehead skin (n = 3) averaged 1.38 (±0.87) ng/mg of wet skin tissue weight. Higher mean baseline levels of PTCA were detected in forearm skin (2.06 ± 0.28 ng/mg wet weight, n = 7). One week after MT-I treatments ended, there was a mean (SD) 49% (±17.6%) increase in forehead skin PTCA levels compared to baseline (P = 0.019, n = 3, by paired sample T-test). The mean (SD) increase in forearm skin PTCA levels was 98% (±25.4%) over the same period (P = 0.003). In contrast, forearm pheomelanin expression following MT-I treatment did not significantly change from baseline. Overall, the MT-I regimen increased the eumelanin:pheomelanin ratio in forearm skin from 51:1 at baseline to 86:1 following MT-I (P = 0.054 by paired sample T-test). These results show that the tanning induced by MT-I in the face and forearm is associated with a significant increase in the eumelanin content of the human skin.