Summary A modified NN medium designated as NNP (Novy, MacNeal and Packchanian) is described for cultivation of trypanosomes in vitro, with special reference to Trypanosoma brucei. Two Uganda strains of Tr. brucei were successfully cultured on NN and NNP media directly from 74 experimentally infected animals during 1954 to 1958. Positive cultures of Tr. brucei were obtained on NNP medium two times from guinea pigs, 24 times from rats, 47 times from house mice (Mus musculus) and one time from American deer mice (Peromyscus maniculatus gambeli). Stock cultures of Tr. brucei have been maintained in vitro on NNP medium by monthly transfer for periods of from 2 months to over 3 years. The flagellates grow readily and luxuriantly not only in the water of condensation but at times on the slant portion of the medium. The morphology of cultural forms of Tr. brucei is characteristic and can at times be differentiated from cultural forms of other species of trypanosomes. Both initial cultures and subsequent subcultures at varying ages grown on the medium described were not infective to experimental animals.