Three hybridoma lines secreting antibodies against human albumin have been characterized. Monoclonal antibody (mAb) A1-01 reacted only with human albumin, whereas antibodies A1-02 and A1-03 reacted with both albumin and transferrin but differed in their affinity for these proteins. The mAbs did not react with albumins or transferrins of other mammalian species. Their specificities were tested by isolating the proteins that bind specifically to the purified immobilized antibodies. Only albumin was isolated on immobilized antibodies A1-01 and A1-02, whereas albumin and transferrin were isolated on antibody A1-03. The minor bands specifically detected by antibody A1-01 were fragments or aggregates of albumin, as shown by two-dimensional electrophoresis and transfer to nitrocellulose sheets. It was found by ELISA that as much as 3 ng/ml human albumin is detectable by mAb A1-01.