Procedures described by previous workers were not satisfactory for the isolation and subsequent maintenance of P. destructor [downy mildew] on onion plants in the greenhouse. High levels of infection and subsequent sporulation were obtained when leaves of onion plants grown from bulbs in a greenhouse were wiped once with a dry pad of cotton immediately prior to inoculation. Sporangia collected from naturally infected onion plants in commercial onion fields in New York, USA (fields sprayed weekly with chlorothalonil and/or mancozeb) were washed 3 or 4 times, suspended in tap water, and used as inoculum. Inoculated plants were incubated in a moist chamber at 14.degree. C for 24 h in the dark, placed in a growth chamber (21,500 lx = 2000 ft-c) at 14.degree. C for 4 days, and then placed on a greenhouse bench with supplemental fluorescent light (16 h) at 18.degree. C for 8 days. Sporulation was induced overnight by placing the infected plants in a moist chamber at 14.degree. C in the dark commencing at 1600-1700 h. Once isolated, the pathogen was maintained by transferring 1-2 day old unwashed sporangia to healthy plants every 3 wk and using the cotton wipe technique and the inoculation and incubation procedures described. All inoculated plants became infected and abundant sporulation was obtained.