To evaluate and characterize the stability of traits conferred by phyA from Aspergillus ficuum, we examined expression of phyA in sexually-derived transgenic cotton progeny and assessed the capacity for phytate-utilization in T4 progeny. The gene (phyA) was expressed only in the roots, but not in the stem and leaf tissues. Phytase activity was 2.38-fold higher in transgenic line L2 than in wild-type (WT) plants. The amount of phosphorus in the leaves was also significantly higher in transgenic lines L2, L6 and L9. Among those lines, L2 performed best, showing increase in phosphorus contents of 10.67% (seedling stage), 8.78% (squaring), 11.77% (flowering) and 11.59% (boll-opening). The level of available phosphorus in the rhizosphere was 19% higher in soil containing transgenic plants compared with the WT. When both transgenic and WT were grown in the same type of soil, the number of bacteria, fungi and actinomycetes was not obviously different. These results indicated that although the T4 generation enhanced the utilization of phytate phosphorus, it had no influence on the number of soil microorganisms. Our findings also demonstrated that phytase-expression lines could be used for developing new varieties of cotton having improved phosphorus uptake from the soil.