The development of Leishmania chagasi, etiologic agent of American visceral leishmaniasis, was studied by light and electron microscopy in the gut of the sand fly, Lutzomyia longipalpis, a natural vector. New aspects of suprapylarian Leishmania behavior were elucidated. In the sand fly midgut, amastigotes transformed into promastigotes (division promastigote I) during a first division sequence within the bloodmeal. Secondary division of these promastigotes resulted in a second form (division promastigote II), and these subsequently elongated into nectomonad promastigotes. Nectomonads existed in long and short populations which divided in the bloodmeal and throughout the midgut lumen after escape from the peritrophic membrane. Nectomonads adhered to the midgut cells in a highly organized manner, with their flagella embedded deep into microvilli and cytoplasm. Migration of parasites from the posterior midgut into the cardia/stomodeal valve region at 36 hr was associated with breakdown of the peritrophic membrane anteriorly. Posterior breakdown at 48 hr resulted in a peritrophic tube open at both ends containing some parasites within the digesting bloodmeal for up to 6 days postinfection. At the stomodeal valve, a myriad of slender and rounded promastigotes attached to the intima by flagellar hemidesmosomes; these may represent a transformation sequence from slender nectomonads to pear-shaped haptomonads. Pear-shaped forms appear to be precursors of paramastigotes, which also attached to the valve intima. Both rounded haptomonads and paramastigotes were found in the esophagus, dividing in a complex sequence initiated by posterior cleavage of the cytoplasm producing unique heart-shaped forms. Dividing paramastigotes also colonized the pharynx up to the cibarial valve. The ultrastructure of paramastigotes suggested that they may be infective forms, capable of some motility in the foregut. Free-swimming "infective" promastigotes were observed throughout the midgut and foregut, were attached in the pharynx (armature region), and were associated with the labrum-epipharynx of the proboscis in 3.6% of flies (16 days). The fine structure of hemidesmosomes in the foregut showed regional specializations, including the presence of plasmalemmar bridges in the gap space.