The type strain of cowpea chlorotic mottle virus (CCMV-T) induces a local and systemic infection in California Blackeye cowpea (Vigna unguiculata (L.) Walp. subs. unguiculata cv. California Blackeye), but accumulates to low levels in inoculated leaves and fails to accumulate systemically in the cowpea plant introduction (PI) 186465. CCMV-R, a mutant strain derived from CCMV-T, accumulates to higher levels than CCMV-T in inoculated leaves and systemically infects PI 186465 plants. The phenotypic determinant of CCMV-R was previously mapped to viral RNA1, but the location of the determinant within RNA1 was not identified. Pseudorecombinants generated from genomic cDNA clones of CCMV-T and CCMV-R indicated that the phenotypic differences on PI 186465 were independent of replication. Through the use of chimeric RNA1 cDNA clones containing portions of CCMV-T and CCMV-R and site-directed mutagenesis, two nucleotides, 299 (amino acid residue 77) and 951 (amino acid residue 294), were identified as being independently critical for the local and systemic accumulation patterns of CCMV-R in PI 186465 plants. A second independently derived CCMV-R-like mutant, identified nucleotide 216 (amino acid residue 49) as being critical for induction of the CCMV-R infection phenotype. Amino acid residues 49, 77, and 294 are within the methytransferase domain of the CCMV 1a protein, suggesting that the methytransferase domain has a role in cell-to-cell and systemic accumulation of the virus that is independent of replication.