Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.