A new technique for conducting site-directed mutagenesis was developed. This method allows the colour selection of mutants through the simultaneous activation or deactivation of the alpha-peptide of beta-galactosidase. Double-stranded DNA plasmids containing large inserts (at least 6.4 kbp in the present experiments) can be used as the mutational template. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. The utility of the method for the analysis of viral genomes was demonstrated by applying it to the mutagenesis of a full-length cDNA copy of RNA-1 of Cowpea mosaic virus (CPMV). Six single-site mutants were initially produced which gave a variety of phenotypes when inoculated on to plants. To confirm that the phenotypes were directly caused by the introduced mutations, a second round of mutagenesis was used to create revertants of two of the mutants. In both cases, the revertants had a wild-type phenotype, demonstrating that the original phenotype was, indeed, the result of the introduced mutation. Overall, the results show that the present technique is a powerful method for site-directed mutagenesis of large DNA fragments, such as whole viral genomes, for functional studies.