The fine structure of metacyclic forms of Trypanosoma brucei propagated in vitro for about 300 days was studied. It was shown that the long and slender parasites possessed a surface coat over the plasma membrane and had many peroxisome-like bodies. The Golgi complex was active and the rough endoplasmic reticulum was prominent. The short and stout parasites originally referred to as "intermediate type" lacked a surface coat, but their cytoplasm contained many lipid inclusions and few peroxisome-like bodies. Like the slender type of parasites, the short and stout ones had very active Golgi complexes and associated vesicles. When these features were compared with those of parasites in infected salivary glands fixed in situ, it was apparent that parasites in the gland canal had similarities with the slender forms cultured in vitro, whereas those found adjacent or attached to the salivary gland epithelium corresponded to the short and stout parasites. Bloodstream forms of the parasite had some features common to the slender parasites grown in culture. These observations suggest that metacyclic trypanosomes cultured in vitro, or those which grow in the tsetse salivary glands, possess the glycerophosphate oxidase enzyme which is probably involved in the utilization of a carbohydrate source.