Opuntia streptacantha and Opuntia megacantha are considered wild species, while Opuntia ficus-indica is deemed a domesticated species. Opuntia species have relevant nutritional and medicinal properties, related to their high levels of phenolics compounds. The objective of this study was to establish callus and cell suspensions of O. streptacantha (cv. Tuna loca), O. megacantha (cv. Rubi reina), and O. ficus-indica (cv. Rojo vigor), and to compare their potential as a source of metabolite production in vitro. Opuntia seeds were physically and chemically scarified obtaining 70-100 % germination. Fast-growing callus and cell suspensions were generated on Murashige and Skoog media supplemented with different concentrations of 2,4-D (0.5, 1.0 mg L-1), and BAP (1.5, 2.0 mg L-1) applied alone or in combination. In general, media containing both, 2,4-D and BAP were more efficient in inducing callus formation than 2,4-D or BAP alone. The content of phenolic acids and flavonoids, and antioxidant activity were all similar in callus and suspensions; however, growth rate was higher in cell suspensions in all species. Production of phenolic acids was correlated to the antioxidant activity, with these parameters being higher in O. streptacantha, the wild species. The concentration of metabolites in callus and cellular suspensions was about three times higher compared with cladodes, indicating that the use of in vitro cultures of Opuntia species is a viable alternative for the production of antioxidants under controlled conditions.