C3a anaphylatoxin derived from the third component of complement has been isolated from rat serum and its complete amino acid seuqence determined. A three-step purification procedure was employed that consisted of gel filtration on Sephadex G-100, followed by chromatography of the anaphylatoxin-containing pool on carboxymethylcellulose. A subsequent separation on DEAE-Sephadex resolved C3a from minor contaminating peptides. Biological studies have shown that purified rat anaphylatoxin is approximately twice as active as human or porcine C3a when tested for smooth-muscle contraction. In addition to the active form of rat anaphylatoxin, a serum carboxypeptidase B inactivated form of C3a (C3ades-Arg) was purified from rat serum and utilized in subsequent structural studies. Sequence analysis of rat C3a was facilitated by a long automated Edman degradation which established the first 55 residues of the anaphylatoxin. Overlapping peptides were generated by cyanogen bromide and trypsin, and the resultant fragments were sequenced by either automated or manual Edman procedures. The primary structure of rat C3a is 70% identical to the previously determined structures of human and porcine anaphylatoxin. Antisera raised to the purified rat peptide do not cross-react immunologically by Ouchterlony analysis with either human or porcine C3a.