Allotypes of the fourth component of complement (C4) can be detected by electrophoresis and immunofixation after treatment of EDTA plasma with neuraminidase (NAse). We have assessed the value of additional treatment with carboxypeptidase B (CPseB). Following treatment with CPseB + NAse, each allele is resolved into a single band, permitting clear definition of overlapping bands seen following treatment with NAse alone. More importantly, C4 allotypes can be determined using stored heparinized plasma or serum. Most C4 null alleles can be assigned without requiring family studies. The approach described is suitable for routine use by tissue typing laboratories.