An immunoradiometric method of the second generation (IR-MA II) is widely used to determine CA125 serum levels. In this study we have evaluated the performance characteristics of a commercially available IRMA CA125 II (Byk-Gulden, Sangtec Diagnostica). The CA125 serum levels were determined in several groups of patients (healthy women, pregnant women, subjects affected by benign and malignant ovarian cancer, patients with liver diseases) with two IRMAs CA125 II (Byk-Gulden, Sangtec Diagnostica and Centocor, Diagnostic Division) and IRMA CA125 I (Byk-Gulden, Sangtec Diagnostica). Our results show a good analytic performance of IRMA CA125 II (Byk-Gulden, Sangtec Diagnostica), a good correlation between IRMAs CA125 II (Byk-Gulden, Sangtec Diagnostica and Centocor, Diagnostic Division), but an unacceptable correlation between IRMAs CA125 II (Byk-Gulden, Sangtec Diagnostica and Centocor, Diagnostic Division) and IRMA CA125 I. A statistically significant difference was observed comparing the values obtained with both IRMAs CA125 II and IRMA CA125 I in the groups of patients. In contrast no statistically significant difference was observed when we compared the values obtained with IRMA CA125 II (Byk-Gulden, Sangtec Diagnostica) and IRMA CA125 II (Centocor, Diagnostic Division). CA125 serum values obtained with the second-generation kits were different from those obtained with the first-generation one; consequently, it is important, especially in the follow-up of cancer patients, that CA125 serum values be obtained with kits of the same generation. Our data seem to suggest the use of second-generation kits to determine CA125 serum levels.