We have isolated and analyzed the DNA encoding the mu heavy chain constant region of a mutant IgM that is defective in initiating complement-dependent cytolysis. By assaying the expression of mu-chain genes that were constructed in vitro from mutant and wild-type gene segments, we have mapped the mutation into a 555-base-pair segment that spans part of the third and fourth constant region domains. In this segment there is one nucleotide change, such that the mutant mu-chain gene encodes asparagine rather than the normal serine at amino acid position 406 in the third constant domain. We have used site-directed mutagenesis to introduce a comparable mutation into the normal mu-chain gene and confirmed that this substitution causes the production of IgM with the original mutant phenotype. Evidence is also provided that the serine-406----asparagine substitution might cause the mutant mu chain to be abnormally glycosylated.