The following research describes the evaluation in the in vitro infection by Leishmania (L.) chagasi in a Lulo cell line, resulting from Lutzomyia longipalpis embryonic tissue, taking into account physicochemical and environmental variables. The medium Gracel/L-15, containing 5% Fetal Bovine Serum (FBS), was used to maintain the cell strain Lulo. The infection process was performed by using Leishmania chagasi CLO44B/84 strain. The physicochemical variables of the growing medium evaluated for the experimental tests of the cell infection were: concentration of FBS (5%, 10%, 15%, and 20%), presence and absence of hemolinfa, pH (5. 5.5, 6. 6.5, and 6.8) and osmolality (35 400 y 450 mOsm/kg). In addition, the temperature which the infected cells were incubated (26°C, 32°C and 37°C) was also evaluated. As substrate for the infected cells, glass cover-slips of 12 mm of diameter in 24 well plates were used. The cover-slips with attached infected cells were removed after 3, 6, and 9 days post-infection. The Experimental observations showed that the greatest percentage of infection (33.3%), was obtained at the 5% FBS level, osmolality of 320 mOsm/Kg, a pH of 6.8 and an optimum temperature of 26°C. By selecting these parameters, high levels of infection were reached and these results will certainly be of great use in future studies and applications of the interaction vector-parasite-cells, in the perspective to contribute with leishmaniasis control.