The objective of this work was to evaluate asepsis protocols, composition and concentration of culture media in the in vitro establishment of three physalis species. In experiment I, seeds of 3 species were used x 3 asepsis protocols. In experiment II, 3 culture media and explants 3 species were used. In experiment III, explants of 2 species x 4 concentrations of MS culture medium were used. In experiment IV, explants of 2 species x 4 sucrose concentrations were used. For experiments II, III and IV after 30 days were evaluated some phytotechnical parameters. The experimental design was completely randomized, in a 3 x 3 (experiments I and II) and 2 x 4 (experiments III and IV) factorial scheme. Protocols II and III were appropriate for P. peruviana germination, and I and III for P. minima, the three protocols were efficient for controlling fungi and bacteria. The MS is the most suitable for the in vitro establishment of three species. The culture medium at 100% concentration obtained better values in the in vitro development parameters evaluated for the species P. peruviana and P. minima. Sucrose concentrations close to 50 and 20 g L -1 favored the establishment of P. peruviana and P. minima.