In vitro formation of Plasmodium berghei ookinetes was studied. Gametocytes produced in vitro were obtained from heart and tail blood of Swiss mice and from blood removed from mosquitoes directly after feeding on these mice. In vitro produced gametocytes were obtained from short-term cultures of the erythrocytic stages of P. berghei. Reproducible ookinete production was obtained in medium RPMI 1640, pH 7.8-8.0, using in vivo and in vitro produced gametocytes. The morphology of developmental stages of ookinetes and degenerate forms at the light microscope level is described. More ookinetes were produced in medium RPMI 1640 compared to MEM and ookinete yield--defined as the ratio between the number of in vitro produced ookinetes/10(5) erythrocytes and the number of exflagellations/10(5) erythrocytes in the infected blood--increased with lower erythrocyte densities in the cultures within the range of dilutions tested. A linear relationship existed between gametocytaemia and the number of ookinetes produced. The methods for in vitro ookinete formation and for estimating ookinete yields enabled us to study aspects of functional maturity of gametocytes independent of mosquitoes. The numbers of exflagellating gametocytes and in vitro ookinete yields in tail blood corresponded with those in heart blood and blood ingested by mosquitoes, suggesting a random distribution of functionally mature gametocytes within the vertebrate host.