The presence of four members of the pyrimidine-specific ribonuclease superfamily was demonstrated in rat liver. Three of them (RL1, RL2 and RL3) were purified and showed ribonuclease activity at pH 7.5 with yeast RNA as substrate. RL1 is identical to rat pancreatic ribonuclease (ribonuclease 1). N-terminal sequence analysis showed the presence of the native protein and several N-terminally degraded components. RL2 and RL3 were N-terminally blocked proteins. After acidic cleavage or CNBr digestion, several parts of their sequences were determined. RL2 has high sequence similarity with neurotoxin-type ribonucleases (ribonucleases 2, 3 and 6). The amino acid sequence of rat liver-type ribonuclease (ribonuclease 4) was determined from a liver cDNA library. It differs at about 20% of the amino acid positions from other mammalian liver-type ribonucleases. The sequence of a peptide of RL3 was identical to that derived from the cDNA sequence of the liver-type ribonuclease. A contaminant of the RL3 fraction had a high sequence similarity with mouse and other mammalian angiogenins. Bovine, porcine and rat liver-type ribonucleases showed a strong preference for poly(U) over poly(C). This preference is a unique property of the liver-type enzymes of the ribonuclease superfamily.