Sporozoites of P. yoelii nigeriensis are 50-100-times more infective to mice than the strain NK65 of P. berghei. To study the mechanisms involved in this striking difference in the infectivity of these closely related species of malaria parasites, we have developed a quantitative PCR targeted to parasite-specific ribosomal RNA. Using this method, we detect RNA from a single sporozoite, and exo-erythorcytic forms of RNA in the livers of mice injected with 200 sporozoites. We find that 20 h after sporozoite injection, there is no significant difference between the amounts of P. berghei and P. yoelii rRNA in the livers of C57/BL6 mice, indicating that these two parasite species invade hepatocytes with similar efficiency. Between 20 and 40 h, however, P. yoelii RNA increases 11 times, while P. berghei RNA increases only 1.6 times. We conclude that the greater infectivity of P. yoelii sporozoites in these mice reflects, at least in part, their superior development in hepatocytes. These data provide for the first time in vivo evidence supporting the notion that species-specificity of malaria is not determined by mechanisms associated with sporozoite attachment and penetration into the hepatocytes.