Traditional culture of Leishmania parasites is labor-intensive and shows poor sensitivity. We evaluated microculture and novel miniculture methods for diagnosis of cutaneous leishmaniasis (CL). Consecutive patients who came to the Leishmaniasis Clinic, Hospital Nacional Cayetano Heredia, Lima, Peru, were enrolled. Lesion aspirates were cultured in traditional tubes containing Novy-MacNeal-Nicolle medium and in miniculture tubes (Eppendorf, Hamburg, Germany) and capillary tubes (microculture) containing RPM11640 medium containing 20% fetal bovine serum. The reference standard was positive results in two of four tests (smear, culture, polymerase chain reaction, or leishmanin skin test). Outcome measures were sensitivity and time to positivity. Fifty-five patients with 74 lesions were enrolled. Of 59 lesions that fulfilled reference criteria for CL, 50 were positive by microculture (sensitivity = 84.7%; P = 0.001), 45 by miniculture (sensitivity = 76.3%; P = 0.042), and 35 by traditional culture (sensitivity = 59.3%). Median time to positivity was three days by microculture and miniculture and five days by traditional culture (P < 0.001). Microculture and miniculture are sensitive and efficient means of diagnosing CL. Copyright © 2008 by The American Society of Tropical Medicine and Hygiene.