Two stocks of Trypanosoma congolense were established in culture at 28 °C using trypanosomes from the proboscides of infective Glossina morsitans. Successful primary cultures were initiated by placing an infected tsetse proboscis beside a bovine dermal collagen explant in Eagle's minimum essential medium supplemented with foetal calf serum. The trypanosomes multiplied rapidly in the medium and also gradually formed an adherent layer on the plastic surface of the culture vessel. Three primary cultures produced organisms infective for mice from 14, 20 and 35 days after initiation and thereafter continuously until days 76, 76 and 52 when they were discarded. Four attempts to initiate infective cultures using infected tsetse proboscides in medium without dermal explants were unsuccessful. When trypanosomes from primary cultures were placed in culture medium with proboscides from uninfected tsetse flies, the parasites multiplied, formed an adherent layer in the culture flasks and were seen in the proboscides within 24 h. A line of 1 stock was serially sub-passaged in this way 4 times during a period of 215 days. Infectivity titrations in mice indicated that primary and sub-passaged cultures each contained similar numbers of infective organisms. Another line of the same stock was also sub-passaged 4 times in medium alone over a period of 186 days. These sub-cultures again retained infectivity for mice, but titrations showed a decrease in infective organism production in the 4th sub-culture. Primary and sub-passaged cultures all included a variety of morphologically different developmental forms of T. congolense, closely resembling those described in the labrum and hypopharynx of Glossina by previous workers. Short metacyclic-like trypanosomes and organisms with proteinaceous surface coats were present in infective cultures. Cultures were successfully re-established after cryopreservation at −196 °C and retained the ability to produce infective organisms.