A 56-nucleotide mutagenic oligomer containing six mismatches with the wild-type template was used to construct multiple transversion mutations in the putative heparin binding region of the rat neural cell adhesion molecule (NCAM) cDNA sequence. Mutants were screened by hybridization to the 56-mer. The relative stability of a mutant DNA:56-mer duplex correlated with the number of base substitutions present in the mutant sequence. Five independent categories of mutants carrying from two to five of the expected nucleotide substitutions were isolated. No mutations other than those directed by the 56-mer were observed. These results suggest a method for generating sets of related predefined substitution mutants.