BACKGROUND: Variant RHCE alleles with diminished expression of C, c, E, and e antigens have been described and indicate the genetic diversity of this gene locus in several populations. In this study the molecular background of variant RhCE antigens identified by standard serologic routine testing in German blood donors and patients was determined.STUDY DESIGN AND METHODS: Samples from blood donors and patients were routinely analyzed for RhCE phenotype using the PK7200 analyzer with two sets of monoclonal anti‐C, ‐c, ‐E, and ‐e reagents. Samples with confirmed variant RhCE antigens were analyzed by nucleotide sequencing of the 10 RHCE exons. A multiplex polymerase chain reaction with sequence‐specific priming (PCR‐SSP) method was established for rapid typing of the rare RHCE alleles.RESULTS: We identified 43 samples with serologic RhCE variants. Molecular analysis revealed variant RHCE alleles in 34 samples. Altogether 22 RHCE alleles were detected; 10 have not been published before. Twenty alleles harbored distinct single‐nucleotide substitutions, 18 of which encoded amino acid changes and 2 of which occurred in noncoding regions. Two samples represented RHCE‐D‐CE hybrid alleles involving different segments of the RHCE Exon 5. A multiplex PCR‐SSP screening for 17 RHCE alleles was negative in 1344 samples of the DNA bank GerBS. The cumulative phenotype frequency was estimated between 1 in 488 (0.20%) and 1 in 8449 (0.012%).CONCLUSION: Single‐amino‐acid substitutions were the molecular basis for variant RhCE antigen expression in most samples. Nucleotide substitutions in RHCE exons were excluded as possible mechanism of diminished RhCE antigen expression in one‐fifth of the serologically identified samples.