Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.