Under nitrogen limitation, Rhizobium meliloti Nod factors induce cell divisions in the inner cortex of alfalfa roots in a still unknown way. These cell division clusters subsequently develop into symbiotically nitrogen-fixing nodules. To study the involvement of plant signals in nodule initiation transgenic alfalfa carrying the promoter of the early nodulin gene MsEnod12A fused to the reporter gene gusA were generated. In untreated plants, low level GUS staining was only found in lateral root primordia and in front of the root apices. After inoculation with R. meliloti or after treatment with purified Nod factors, GUS activity was first induced in the cell division foci of the inner cortex. The GUS staining patterns in nodules and roots were in agreement with the activation of the endogenous MsEnod12A gene as revealed by reverse transcription-PCR analysis, rendering the MsEnod12A-gusA fusion a valuable novel marker for studying the onset of nodule and lateral root developmental processes. Treatment of roots with purified Nod factors and cytokinins induced similar patterns of cortical cell division, GUS staining and amyloplast accumulation while upon application of auxin transport inhibitors and auxins these patterns were different. Like the Nod factor responses, the cytokinin responses required photosynthesis and limiting combined nitrogen supply. Thus, cytokinins and Nod factors may share elements of their signal transduction pathways to the inner root cortex. A model on the possible involvement of cytokinins in coordinating plant metabolism with nodule initiation is proposed.