The highly appreciated Euvitis subgenera species (2n=38) are very susceptible to pests and diseases. Tolerance/resistance may be found in the closely related Vitis rotundifolia cultivars (2n=40), but the poor rooting characteristic of this species is a problem, and conventional crossings between Euvitis and V. rotundifolia are complicated because of different chromosome numbers. Therefore, somatic hybridization may be an alternative for gene transference between these species. The establishment of an efficient in vitro procedure may facilitate future genetic manipulations. Furthermore, in vitro success may be an indicative of protoplast totipotency. The goal of this research was to test 11 cultivars from different species for their in vitro cultivation and protoplast isolation capacity. Different doses of benzyladenine (BA) were tested and explants were cultivated in both Lloyd and McCown's Woody Plant Medium (WPM) and Murashige and Skoog medium (MS). We established an efficient in vitro procedure and plants of C. sauvignon, Syrah, SV 12-375, Scuppernong, Magnolia, Higgens and B. beauty were regenerated. No rooting problem was observed in vitro. Black spanish and Herbemont callus were kept in vitro, but plants were not regenerated. SV-12327 and Jumbo died. WPM was more efficient than MS for most cultivars. The V. vinifera cultivars C. sauvignon and Syrah developed well in both media. Protoplast isolation was more efficient using leaves rather than callus or suspension cells. BA at 3 μM·L-1 induced organogenesis while 10 μM·L-1 induced callogenesis except for Syrah, where 1 μM·L-1 induced organogenesis. Protoplasts were isolated from Herbemont and C. sauvignon and microcallus were obtained.