This study was conducted to evaluate the effects of proline and glutamine on in vitro callus induction and subsequent regeneration and to develop a reproducible and highly efficient plant regeneration protocol in four rice genotypes, viz. Pawana, Jaya, Indrayani and Ambemohar. Considerable variation in response to plant growth regulators and amino acid supplements used was observed in all the four genotypes. Medium supplemented with proline and glutamine was shown to be superior to medium without proline and glutamine. The best callusing from mature embryo was observed on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 500 mg/L proline and 500 mg/L glutamine. Shoot induction was higher in the callus obtained from medium supplemented with 500 mg/L proline and 500 mg/L glutamine. The highest shoot regeneration frequency (83.2%) was observed on MS medium with 2.0 mg/L benzylaminopurine, 0.5 mg/L 1-naphthaleneacetic acid, 500 mg/L proline, and 500 mg/L glutamine in the callus obtained from MS medium supplemented with 2.0 mg/L 2,4-D, 500 mg/L proline and 500 mg/L glutamine. Among the four genotypes, Pawana has the highest regeneration efficiency (83.2%), whereas the regeneration efficiency of the rest three rice genotypes was in the range of 32.0% to 72.3%. This optimized regeneration protocol can be efficiently used for Agrobacterium mediated genetic transformation in rice.