Sexual differentiation is essential for the transmission of Plasmodium to mosquitoes and therefore, for the spread of malaria. The molecular mechanisms underlying sexual differentiation are poorly understood but may be elucidated by a detailed study of the regulation of expression of sexual stage specific genes. In the present work we describe the differential expression of the gene encoding the sexual stage specific protein, Pfs16. We have conducted a comparative analysis of pfs16 promoter activity, RNA levels and the rate of de novo protein synthesis during development of Plasmodium falciparum. Furthermore, we have determined the pattern of expression of pfs16 transcripts at the single cell level by in situ hybridisation. We show that the expression of pfs16 is induced immediately following the invasion of a red blood cell in sexually committed ring stage parasites and continues throughout gametocytogenesis and in macrogametes. The expression of pfs16 is regulated at the level of transcription initiation and modulated by a post-transcriptional process. These results demonstrate that the expression of the pfs16 gene is the earliest event in the sexual differentiation process of P. falciparum described to date.