This study evaluated the significance of serum pancreatitis-associated protein (PAP) assay, as a marker of hepatocellular carcinoma (HCC), in comparison and combined with alpha-fetoprotein (AFP) assay. Sixty-five patients with HCC, 59 with liver cirrhosis (LC) and 68 asymptomatic controls (C) were studied. PAP and AFP values significantly increased from C to LC and HCC group (p < 0.0001). The area under receiver-operating characteristic (ROC) curve for the two markers was not statistically different. At 100% specificity, ROC analysis gave a cut-off level for AFP of 166 IU/l with 40% sensitivity, and a cut-off level of 240 microg/l for PAP with 23% sensitivity. Diagnostic accuracy of combined AFP and PAP assay was significantly higher than AFP alone. Sensitivity according to tumor size also improved using the combined assay, especially for tumors <5 cm. Stepwise logistic regression indicated that AFP, but not PAP, was associated with an increased risk of developing HCC. These data confirm that PAP production is increased only in some cases of HCC and that the combined PAP and AFP assays do not significantly improve specificity over AFP assay alone. Consequently, PAP assay can only be recommended in cases of justified suspicion of HCC with negative AFP.