The subclinical and clinical Plasmodium elongatum and Plasmodium relictum infections of captive-reared African black-footed penguins (Spheniscus demersus) were evaluated in nine adult and 29 juvenile penguins in the Baltimore Zoo (Maryland, USA) during summer 1988 and winter 1989. Two diagnostic methods were used: Giemsa-stained thin blood films, and subinoculation of penguin blood into 1-day-old Peking ducklings. Chloroquine and primaquine treatment was applied to all parasitemic juvenile penguins. Twenty-nine parasite-free, juvenile penguins were monitored for parasitemia by Giemsa-stained thin blood films every two weeks for 26 weeks of their first outdoor exposure. Eighteen of 29 penguins experienced naturally acquired malaria; 14 were infected with P. elongatum, three with P. relictum, and one bird had a mixed P. relictum and P. elongatum infection. Eleven of 18 juveniles became parasitemic again after chloroquine and primaquine treatments. Based on Giemsa-stained thin blood smears and sub-inoculation of penguin blood into 1-day-old ducklings, performed in a mosquito-free environment in winter, nine adult penguins had no evidence of Plasmodium spp. infection. After dexmethasone-induced immunosuppression, four of six of these nonparasitemic adult penguins were found to be infected with P. relictum by the blood inoculation method.