Early stages in the infection of leguminous plants by Rhizobium spp. are restricted at low pH and are further influenced by the presence of Ca and Al ions. In the experiments reported here, transcriptional and translational fusions of the Escherichia coli lacZ gene to Rhizobium leguminosarum biovar trifolii nodulation (nod) genes were used to investigate the effects of pH and of Ca and Al ions on nod gene expression. The regulatory nodD gene in R. leguminosarum biovar trifolii was constitutively expressed at a range of pH levels between 4.8 and 6.5, and expression was not affected by the addition of 22.5 muM Al or 1,250 muM Ca. Induction of expression of nodA, nodF, and region II nodulation genes in the presence of 5 x 10 M 7,4'-dihydroxyflavone was restricted at a pH of <5.7 and was extremely poor at pH 4.8. Induction of nodA expression was further restricted by 22.5 muM Al over a range of pH levels but was increased in the presence of Ca. The addition of Ca, however, only slightly alleviated the Al-mediated inhibition of nodA induction. Induction of expression of nodA was equally sensitive to low pH in three strains of R. leguminosarum biovar trifolii (ANU845, ANU815, and ANU1184), which exhibited contrasting growth abilities in solution culture at a pH of <5.0. Aluminum, however, differentially affected the induction of nodA in these three strains, with the most Al-tolerant strain for growth being the most Al-sensitive strain for nod gene induction. Poor induction of expression of nodulation genes in R. leguminosarum biovar trifolii was considered to be an important factor contributing to the acid-sensitive step of legume root infection.