Three linear antigenic regions on the N protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were identified by using peptides which reacted in ELISA with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the protein: Thrll to Gly30 (N3 peptide), Ser231 to Ala~50 (N25 peptide) and Thr3v 1 to Leu391 (N39 peptide). The site represented by the N39 peptide reacted with four subgroup A-specific MAbs. There were minor variations in the amino acid epitope dependencies of each of these MAbs. Two additional antigenic regions Serl~ 1 to Arg150 and Alals ~ to Leu.~00, were represented by peptides that reacted with human convalescent sera, but these peptides did not differentiate between acute and convalescent sera from RSV-infected humans. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the N protein from a nucleocapsidcontaining homogenate derived from extracts of RSV-(subgroup A and/or B)-infected 35S-labelled cells in a radioimmuneprecipitation assay (RIPA); the sera were also used to demonstrate RSV infection in cells by immunofluorescent assay (IFA). Anti-N3 peptide sera precipitated N41, the full-length (M r 41000) form of N protein, in a RIPA done on a soluble protein pool. Anti-N39 (C-terminal region) peptide sera precipitated both forms, suggesting that N3s (M r 38 000) is an N-terminally truncated (probably at position Tyr23 located inside the N3 peptide linear antigenic region) form of N~l protein.