This study was conducted to investigate sporulation of Leptosphaerulina trifolii (Rost.) Petrak and to improve infection of white clover plants during experimental screening of germplasm for resistance to this ascomycete. Sporulation at 23 ± 2°C was greatest after 9 ± 1 days growth on V-8 juice agar under fluorescent and near-ultraviolet lights. Infection was greatly promoted by keeping leaf surfaces wet during the initial period of ascospore deposition. Increasing the period of ascospore deposition beyond 12 h did not significantly increase plant infection. The number of pepper spot lesions which appeared on leaves was significantly correlated with the number of ascospores released by sporulating cultures suspended over plants during the spore deposition period. More leaf spots were produced when the temperature was 25°C during ascospore deposition and 20°C during incubation than when a regime of 20/15°C was used.