Atypically high radioimmunoassay estimates of erythropoietin in serum samples from two out of a series of 130 normal Norwegian children have been found to be invalid. Invalidity was due to non-parallelism of radioimmunoassay (RIA) response dilution lines given by these two sera when compared with the second International Reference Preparation (IRP). In contrast, immunoreactive erythropoietin in other sera usually shows parallelism with the IRP. Fractionation by gel filtration of the sera showing non-parallelism in the RIA showed them to contain an erythropoietin cross-reactant which was eluted before both an 125I recombinant human erythropoietin marker and normal erythropoietin. In family studies, non-parallelism of RIA response dilution lines and the large molecular size erythropoietin cross-reactant was found in serum from one sibling but not in five other family members of one of the two index cases. Radioimmunoassays based on two different antisera differed in recognition of the abnormal cross-reactant of erythropoietin.