a institute of Soil and environmental Sciences, university of agriculture, Faisalabad, Pakistan; b Physiology/chemistry Section, central cotton research institute, Multan, Pakistan; c department of agronomy, university of agriculture, Faisalabad, Pakistan ABSTRACT Transgenic Bt-cotton produces Bt-toxins (Cry proteins) which may accumulate and persist in soil due to their binding ability on soil components. In the present study, the potential impacts of Btand non-Bt genotypes of cotton on soil microbial activity, substrate use efficiency, viable microbial population counts, and nutrient dynamics were studied. Two transgenic Bt-cotton genotypes (CIM-602 CIM-599) expressing cry1 Ac gene and two non-Bt cotton genotypes (CIM-573 and CIM-591) were used to evaluate their impact on biological and chemical properties of soil across the four locations in Punjab. Field trials were conducted at four locations (Central Cotton Research Institute-Multan, Naseer Pur, Kot Lal Shah, and Cotton Research Station-Bahawalpur) of different agro-ecological zones of Punjab. Rhizosphere soil samples were collected by following standard procedure from these selected locations. Results reveled that Bt-cotton had no adverse effect on microbial population (viable counts) and enzymatic activity of rhizosphere soil. Bacterial population was more in Bt-cotton rhizosphere than that of non-Bt cotton rhizosphere at all locations. Phosphatase, dehydrogenase, and oxidative metabolism of rhizosphere soil were more in Bt-cotton genotypes compared with non-Bt cotton genotypes. Cation exchange capacity, total nitrogen, extractable phosphorous, extractable potassium, active carbon, Fe and Zn contents were higher in rhizosphere of Bt-cotton genotypes compared with non-Bt cotton genotypes. It can be concluded from present study that the cultivation of Bt-cotton expressing cry1 Ac had apparently no negative effect on metabolic, microbiological activities, and nutrient dynamics of soils. Further work is needed to investigate the potential impacts of Bt-cotton on ecology of soil-dwelling insects and invertebrates before its recommendation for extensive cultivation.