The effect of various concentrations of thidiazuron (TDZ) with or without 2.7 μM of α-naphthalene acetic acid (NAA) on adventitious shoot formation of two Petunia hybrida cultivars was studied. Seeds from ‘Daddy Blue’ and ‘Dreams White’ cultivars were germinated in vitro. Expanded leaves from both seedlings and regenerated shoots were taken as explants. Explants were placed on MS media supplied with different concentrations and combinations of the growth regulators. Cultures were incubated under dark conditions for three weeks and then moved under light condition. The ability to form shoots varied depending on the growth regulator combination. The highest shoot percentage (87.5 to 90%) with both explant sources was obtained when TDZ was used at levels between 0.5 and 8 µM in both cultivars. Regeneration was reduced significantly when TDZ was combined with 2.7 μM NAA. A similar trend of average number of shoots produced was observed.