Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17 (BTV-17) were investigated with a panel of five neutralizing monoclonal antibodies (MAbs). One MAb (MAb 034) was originally raised to BTV serotype 10 (BTV-10) but also neutralizes BTV-17 (P. V. Rossitto, and N. J. MacLachlan, 1992, J. Gen. Virol. 73, 1947-1952). Competitive binding studies indicate that the MAbs recognize at least two epitopes on the neutralizing outer capsid protein VP2 of BTV-17. The MAbs were used to select neutralization-resistant variant [escape mutant (EM)] viruses and to determine the phenotypic characteristics of these EM viruses by immunoprecipitation and neutralization assays. Sequencing of the L2 gene, which encodes VP2, identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. Four amino acids in three regions of VP2 are critical to the expression of the epitopes recognized by the panel of neutralizing MAbs. Amino acid 199 affects the binding of MAbs 17.82, 17.83, and 17.813; amino acid 213 affects the binding of MAb 17.85; and amino acids 327 and 582 synergistically affect the binding of MAb 034. Similarly, amino acids 327 and 402 synergistically affect the binding of MAb 034 to BTV-10 (C. D. DeMaula, H. W. Heidner, P. V. Rossitto, C. M. Pierce, and N. J. MacLachlan, 1993, Virology 195, 292-296), suggesting that the neutralizing epitope common to BTV-10 and BTV-17 has a similar location in VP2 of these two antigenically distinct viruses.