Pigeon pea ( Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts- USDA191 and USDA110-and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both USDA191 and a T3SS mutant RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with USDA110 and Δ136 (a T3SS mutant) resulted in the formation of Fix- and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in USDA110 nodules, while it was abundantly present in Δ136 nodules. Results of competitive nodulation assays indicated that Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of , due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.