In contrast to the findings of Rawlings et al.. Of additional significance is the lack of expression of Pfg377 transcripts and protein prior to Stage IV of gametocyte development. Thus any technique which estimated sex ratio based on Pfg377 transcript or protein abundance would deliver an “apparent” sex ratio that was extremely male biased prior to stage IV. As commitment of any particular P. falciparum parasite to male or female gametocyte development occurs in the parental asexual parasite, this apparent ratio is an artefact of the markers chosen, as a-tubulin II is expressed by both sexes in these early stages, and female gametocytes do not produce reliably detectable levels of Pfg377 until they reach stage IV. Further, any contamination of mature gametocyte preparations with earlier stages, due to inadequate synchronisation, will also produce artefactually malebiased sex ratio estimates. The development of antibody-based fluorescent staining protocols for male- and female-specific proteins expressed earlier in gametocyte development would overcome this problem. Drew and Reece developed a qRT-PCR assay for the quantification of sex ratios in P. chabaudi, however the BAY-60-7550 extended maturation of P. falciparum gametocytes over many days means the relative abundance of sex-specific mRNA through development must be characterised prior to calibration of such assays against microscopic data. Indeed, it may be necessary to quantify transcript abundance of more than one male- and female-specific gene in order to correct for maturity in estimating sex-ratio. This important effect of maturity is unique to gametocyte development in P. falciparum, among the malaria parasite species that can be studied in the laboratory. The ability to rapidly and accurately derive estimates of gametocyte sex ratio provides a valuable additional phenotype for studies of the in vitro effect of antimalarial compounds on parasite transmission potential. Enumeration of gametocytes in toto may fail to recognise important transmission-blocking effects which only affect one sex, particularly if these were male gametocytes; loss of all males would completely prevent transmission to mosquitoes, but cause only a small decrease in total gametocyte numbers if females were unaffected. The ideal method for routine evaluation of drug effects is unlikely to be IFAT, as it is difficult to scale-up for high-throughput assays, but our antibodybased methods could be adapted to flow cytometrics. Further, we suggest that the approach we have described is ideal for comparative studies of different gametocyte-producing lines, phenotypic studies of transgenic gametocyte lines following targeted gene-disruption, and, most importantly, for validation of high-throughput methods.