Zoospores are major dispersal and infective propagules of pythiaceous species. Built upon a recently developed 'wet-plate' method, the objectives of this study were to develop a better understanding about zoospore production biology. Four broth media and five incubation temperatures were evaluated with 12 isolates of Phytophthora nicotianae and 17 other pythiaceous species in this study. The 'wet-plate' method worked the best for heterothallic species, especially those isolates that do not produce chlamydospores. These species included Phytophthora citrophthora, P. nicotianae, Phytophthora palmivora and Phytophthora tropicalis. They readily produced 10 5 -10 6 zoospores/ml. Overall, most species and isolates produced more zoospores with 20% clarified V8 broth than the other three media: rye, lima bean and carrot. The optimal temperature for nutrient-deprived culture without free-flowing water to produce sporangia typically is 5°C cooler than that for vegetative growth. Fresh and revived cultures are more prolific than those that had been subcultured multiple times. These findings will assist oomycete researchers, adding quality, productivity and efficiency to their future zoospore-based studies.