To investigate the mechanisms regulating the stage specific expression of the Plasmodium knowlesi circumsporozoite (CS) antigen gene, the sporozoite genomic DNA copy of the CS gene has been isolated and compared with the blood stage genomic DNA and the sporozoite cDNA copies. The genomic DNA sequences of the two developmental stages are identical across 4 kilobase pairs of the chromosome containing the entire CS gene transcriptional unit. From restriction enzyme mapping no DNA rearrangements over 15 kilobase pairs of the chromosome containing the CS gene appear to be involved in its stage-specific expression and its regulation appears to be at the level of transcription or RNA stability. S1 nuclease and primer extension transcript mapping studies suggest that the CS mRNA has multiple start sites, that the leader sequence is devoid of introns, and is approximately 270 bases long. Consensus eukaryotic TATA and CAAT box sequences and potential regulatory elements, including sequences highly homologous to the reiterated and core enhancer sequences of SV40 precede the gene.