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The bdhA-xdhA2-xdhB2 mixed-function operon was used to demonstrate the application of crossover PCR to the construction of in-frame, non-polar deletion-insertion mutations in Sinorhizobium meliloti. Replacement of a 474-bp internal portion of the 774-bp coding sequence of bdhA with a 21-bp in-frame synthetic sequence resulted in loss of the bdhA-encoded d-3-hydroxybutyrate dehydrogenase activity. Such mutants retained the xanthine oxidase activity encoded by xdhA2-xdhB2, thus illustrating the non-polar nature of the mutation. This method of constructing unmarked, in-frame deletions should be generally applicable to functional genomics studies in S. meliloti and other α-Proteobacteria.