We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax® on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax® led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID₅₀) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID₅₀ obtained for CD-1 mice (147 sporozoites) was significantly higher.