Rhizobium etli elicits nitrogen-fixing nodules on the roots of Phaseolus vulgaris. Using a composite dual-marker mini-Tn5 transposon carrying combinations of a constitutively expressed gfp gene and a promoterless gusA gene, we identified novel genes required for an efficient symbiosis. The induction of the gusA gene was used to determine the expression level of the different target genes under conditions partly mimicking the symbiotic environment ex planta. The green fluorescence was used to localize the bacteria in infection threads or inside the plant cells. Among the identified R. etli mutants, several produced a Nod- phenotype, whereas others were Fix- or displayed a reduced acetylene reduction activity during symbiosis. Partial sequence analysis of the mutated genes allowed us to classify them as nodulation genes, nitrogen fixation genes, genes possessing various enzymatic functions previously not yet associated with symbiosis, and genes displaying no similarity to any other sequence in the database. This methodology can be used to screen large numbers of mutants in the search for novel genes important for Rhizobium-legume symbiosis, and may be adapted to study other plant-bacterium interactions.