A new commercially available enzyme-linked immunosorbent assay (ELISA) kit has been evaluated for the measurement of neopterin concentrations in serum, plasma and urine. This competitive ELISA is technically simple, requires only small sample volume and is rapid to perform. The assay procedure consists of sequential 1.5 h and 10 min room temperature incubation steps. The ELISA is accurate, sensitive, specific, and precise. Linear regression analysis of neopterin concentrations measured with the new ELISA and with an established method yielded a highly significant correlation (r = 0.99). The new assay is applicable to ELISA workstations, thus enabling determination of neopterin in large series of samples. The neopterin ELISA kit has been used in routine laboratory testing of blood donations in a blood bank.