Clones of single oocysts, single sporocysts, single sporozoites, single proliferative parasites, single cysts and single cystozoites of Isospora (Toxoplasma) gondii (KB-strain) were made under visual control using a de Fonbrune micromanipulator. Cloning was successful in 28, 32, 21, 8, 54 and 7% of the trials, respectively. All clones were used for monoclonal infection in non-immune conventionally reared (CV) or specified pathogen-free (SPF) cats. Pre-patent and patent periods, sporulation percentages of excreted oocysts, mouse infectivity of sporulated oocysts, antibody response and immunity to reinfection of CV cats were determined. For these parameters almost no differences were observed between monoclonal infections and infections described with the non-cloned KB-strain. In all cats autopsied during the patent period, 5–8 days post-infection, macrogametes, microgametes and oocysts were found. Since meiosis occurs during sporulation and since all free-living and intermediate host stages proved to be bisexual, it is concluded that sex differentiation in I. (T) gondii is not determined by segregation of sex chromosomes or sex genes but is caused by some final host factor(s) that induce(s) differential gene expression in genetically identical cells.