BACKGROUND In this study we analysed the reliability of HPLC determination of 2.3.5-pyrroletricarboxylic acid (PTCA). This product derives from oxidation of melanin in human hair, and is a good candidate as a risk marker for skin tumors. METHODS We determined PTCA in 100 melanoma cases and 100 controls, 21 replicates from six different reference hairs, two trace elements, and one reference sample (brown hair). RESULTS Work-up procedures showed an almost perfect reproducibility with an Intraclass Correlation Coefficient (ICC) of 0.990. We noticed a low, detectable, but not statistically significant decrease in reproducibility proportional to the amount of PTCA. Agreement between determination following injection of the same solution in HPLC column was also high, with an overall ICC of 0.986. Simultaneous analysis of reproducibility showed a partial ICC for work-up (0.986), for injection (0.987), and an overall standardised ICC (0.975). The analysis of the two reference tracers in successive tests showed a weak, not statistically significant, decreasing linear drift, possibly due to various factors, such as aging of chemical solutions and HPLC columns. CONCLUSIONS PTCA extracted from human hair through oxidation and determined with HPLC can be considered a reliable marker as a candidate for identifying persons at high risk for melanoma.