The effect of different concentrations of hormones on callus induction in tomato was evaluated, at Agricultural Biotechnology Institute (ABI), NARC, Islamabad in 2002. Two varieties ‘RIO’ and ‘Roma’ were tested for callus induction on MS (Murashige Skoog) medium containing several combinations of IAA (Indole acetic acid), and Kinetin, while 2,4-D (2,4-Dichlorophenoxi acetic acid) used alone. Using cotyledons as an explant source the highest callus response was recorded for Roma (94.48%) followed by Rio (92.15%), while using leaf segments as an explant source the highest callus response was observed in Rio (89.11%) followed by Roma (88.87%), on the medium containing 0.2mg/1 IAA and 2mg/1 kinetin. The least response, 25.33% for cotyledon and 20.13% for leaf explants, were found with 2,4-D used alone as an auxin sourve for Roma variety, whereas in Rio least frequencies (36.6 and 33.3%) of callus formation were observed on the medium containing 0.1mg/1 IAA and 0.1mg/1 Kinetin when both types of explant were cultured. So, the medium containing 0.2 mg/1 IAA and 2mg/1 Kinetin was proved to be optimal for callus formation in tomato. Cotyledons of Roma were more responsive for callus induction as compare to cotyledons of Rio while leaf segments of Rio gave highest callus than Roma’s leaf segments.