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The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and Borreliella spirochetes are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick-or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.