Sexuality in the oomycete Plasmopara viticola, the causal agent of grapevine downy mildew, was studied using isolates from five populations from North America and Europe. Leaf discs of Vitis vinifera cv. ‘Chardonnay’ were inoculated with either individual single-sporangiophore isolates, or in all possible pairwise combinations of 25 isolates from New York State, USA. The occurrence of oospores in leaf discs indicated that the pathogen was heterothallic with two mating types, P1 and P2 in a ratio of 11 : 14 for this population. Heterothallism was confirmed when three representative isolates of each mating type from New York were coinoculated with each of 40 isolates from populations of P. viticola from Michigan, Missouri (USA), Germany and Italy. For each isolate tested, oospores formed with either test isolates of P1 or test isolates of P2 mating types, indicating that the isolates were exclusively P1 or P2 only. For these same isolates, no oospores formed as a result of self-crosses. The ratio of P1 : P2 mating types for all isolates in the study was 27 : 38, statistically equivalent to a 1 : 1 ratio according to χ2 analysis (P = 0·68).