Mutants of Rhizobium trifolii strain 7012 defective in C(4)-dicarboxylate transport were isolated by using a selective procedure based on pH indicator media. The mutant strains CR7098 and CR7099 failed to grow on or transport succinate, fumarate, or malate, but grew at wild-type rates on several other carbon sources. The C(4)-dicarboxylate transport system was inducible in strain 7012, but was expressed constitutively in four out of five succinate-positive revertants of strain CR7098. In the fifth CR7098 revertant (strain CR8008) the system was inducible. However, in contrast to strain 7012, strain CR8008 failed to use the C(4)-dicarboxylates in the presence of a second carbon source. Revertants of strain CR7099 were similar to strain 7012. Both strains CR7098 and CR7099 nodulated white and red clover at a rate similar to that of strain 7012, but nodules formed by the mutant strains were white and ineffective. Microscopic examination showed that the pattern of development of white clover nodules formed by strain CR7098 was similar to that observed with nodules formed by strain 7012, except that large amounts of starch accumulated in bacteroid-filled cells and senescence occurred earlier. Revertant strains were effective, except for strain CR8008, which formed ineffective nodules. The results show that a supply of C(4)-dicarboxylates to bacteroids is essential for nitrogen fixation in clover nodules. However, rhizobia within plant cells must also utilize other carbon sources to support growth and division.