The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.