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Only three species of African freshwater fish trypanosomes are currently recognised as valid. These are Trypanosoma toddi Bouet, 1909, T. mukasai Hoare, 1932 and T. tobeyi Dias, 1952. mainly from East African hosts. Only T. mukasai has previously been reported from southern Africa, including South Africa and Botswana. To date, no fish trypanosomes from the Zambezi River have been described. During September 2004 we had the opportunity to study blood smears of two Zambezi grunters, Parauchenoglanis ngamensis (Boulenger 1911) and 13 Spotted squeakers, Synodontis nigromaculatus, Boulenger, 1905, caught with baited hooks in the Zambezi River. Fish were identified, measured (total length) and anaesthetised with phenoxy-ethanol. Blood was drawn from the caudal vein of anaesthetised fish with a 25G-needle attached to a 1-ml syringe and smeared on clean glass microscope slides. Blood smears were fixed for 5 minutes in absolute methanol and subsequently stained for 25 minutes in phosphate-buffered Giemsa stain (pH 6.8). Stained smears were screened for the presence of trypanosomes with the aid of an Olympus compound microscope. All trypanosomes detected were digitally photographed with a Zeiss Axioplan 2-photomicroscope. Digitally stored images of the trypanosomes were measured with Axio Vision software following standard methods. These measurements included mid-nucleus to anterior (MA), mid-nucleus to posterior (MP), mid-nucleus to kinetoplast (MK), posterior to kinetoplast (PK), nucleus length (NL), body width (LW), total body length (TL), general body length including free flagellum (AL). The nucleur index (NI = MP/MA) as well as the free flagellum index (F1 = (AL-TL)/TL) was calculated. Both the Zambezi grunters (100%) and 7 of the 13 spotted squeakers (54%) were infected with trypanosomes that conformed to the morphometric and morphological descriptions of T. mukasai. In captivity there was a decline in the number of trypanosomes in the blood of the Zambezi grunters and no parasites were present after 10 months. This is the first record of fish trypanosomes from the Upper Zambezi River and the first record of their retention in captivity in the absence of a vector. [ABSTRACT FROM AUTHOR]