Invertases hydrolyze disaccharide-sucrose into glucose and fructose, and are involved in various metabolic processes in plants as key modulators of source/sink activities. Cotyledons of Vigna radiata seeds are photosynthetically inactive sink tissues that store sucrose. However, a sink to source transition occurs when de-embryonated cotyledons are grown under in vitro conditions to undergo differentiation. In this study, two cotyledon types, Cot and Cot E based on a unique differential regeneration response reported earlier, are grown separately in vitro on Gamborg's B5 basal nutrient medium (Gamborg et al. 1968), 2% sucrose, and supplemented with 5, 10, 15, or 20 μM N6-benzyladenine (BA). Temporal differences in acid invertase enzyme activity are observed in differentiating explants. In general all BA levels and both in the presence and/or absence of sucrose, acid invertase levels are lower in Cot than Cot E explants. A higher frequency of regeneration of Cot E explants is positively correlated with higher invertase activity. A partial acid invertase (VrAI1) gene has been isolated, cloned, and sequenced (GenBank ID. GQ227550). Using RT-PCR, temporal variations in VrAI1 gene expression are observed in differentiating cotyledons and the transcripts are higher in Cot E than in Cot explants, up to 9 days following culture. Moreover, invertase gene expression is highest in Cot explants at 12 and 15 days following culture. Thus, temporal variations in both amounts of transcripts and in enzyme activity between these two explant types correspond to in vitro responses to shoot differentiation. It is suggested that acid invertase may be involved in temporal differential regeneration patterns observed in Cot and Cot E explants of V. radiata. © 2011 Springer Science+Business Media B.V.