The diagnosis of spotted fever is based on characteristic signs and symptoms but requires laboratorial confirmation because of the possible differential diagnosis from other diseases like leptospirosis, enterovirus, meningococcemia and typhoid fever. Laboratorial confirmation may be done by detection of specific antibodies which is possible only 5-10 days after the onset of the symptoms or by the isolation of Rickettsiae from blood and/or skin biopsy and from ticks collected in the patient or in the animal reservoir. The isolation of Rickettsiae from blood or skin biopsy results in an early diagnosis of spotted fever since in the rickettsiemic phase of the disease there is no detectable level of antibodies in the serum. With the purpose of facilitating the diagnosis of Spotted Fever we have standardized the isolation of Rickettsiae in cell culture by a method that is less time consuming and that reduces the biological risks than isolation in guinea pigs. Vero cell cultures were inoculated with the Sheyla Smith strain of Rickettsia rickettsii provided by CDC (Atlanta-USA). The identification was performed by indirect immunofluorescence technique. The presence of green fluorescent organisms characterized the growth of the agent. Ulterior confirmation of the methodology was done by isolation of the spotted fever agent from skin biopsy of a patient from an endemic area and from Amblyomma ticks that are the reservoir and vector of the Brazilian spotted fever.