Bartonellosis is an important public health problem in medically underserved villages of the Peruvian Andes. The causative agent, Bartonella bacilliformis, is transmitted by bites of phlebotomine sand flies, genus Lutzomyia. From February 2002 through June 2004 phlebotomine sand flies were sampled two days and nights per month at case sites in each of three villages in an epidemic focus of bartonellosis in the Vilcanota Valley, south of Cusco, Peru. Over 15,000 sand flies were captured with mouth aspirators from resting sites inside and outside of houses in the early morning and with CDC light traps during the night. Over 80% of the flies were captured indoors. Except for fewer than 50 Warileya spp., all sand flies were identified as Lutzomyia peruensis, the suspected vector of Bartonella bacilliformis in this region. Analysis of collection data over time shows a unimodal annual pattern related to high minimum nighttime temperature and rainfall. Sand fly populations are at lowest densities in June and July, corresponding to the “winter” dry season, when day and night time temperatures and relative humidity are lowest. As temperatures rise in August, sand fly populations increase to a pronounced peak in November, prior to onset of the “summer” (December) rainy season. Once the rains begin and temperatures drop, sand fly populations decline steadily through July. Nightly sand fly activity is limited to early evening (1800-2100 hrs) from March through July. As nighttime temperatures increase in late August through November, activity extends throughout the night.