Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics in vitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promastigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesis in vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression in vitro, also showed selective expression by promastigotes in the fly, and was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut.