The AGC protein kinase family (cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases C) have important roles regulating growth and development in animals and fungi. They are activated via lipid second messengers by 3-phosphoinositide-dependent protein kinase coupling lipid signals to phosphorylation of the AGC kinases. These phosphorylate downstream signal transduction protein targets. AGC kinases are becoming better studied in plants, especially in Arabidopsis (Arabidopsis thaliana), where specific AGC kinases have been shown to have key roles in regulating growth signal pathways. We report here the isolation and characterization of the first AGC kinase gene identified in Medicago truncatula, MtIRE. It was cloned by homology with the Arabidopsis INCOMPLETE ROOT HAIR ELONGATION (IRE) gene. Semiquantitative reverse transcription-polymerase chain reaction analysis shows that, unlike its Arabidopsis counterpart, MtIRE is not expressed in uninoculated roots, but is expressed in root systems that have been inoculated with Sinorhizobium meliloti and are developing root nodules. MtIRE expression is also found in flowers. Expression analysis of a time course of nodule development and of nodulating root systems of many Medicago nodulation mutants shows MtIRE expression correlates with infected cell maturation during nodule development. During the course of these experiments, nine Medicago nodulation mutants, including sli and dnf1 to 7 mutants, were evaluated for the first time for their microscopic nodule phenotype using S. meliloti constitutively expressing lacZ. Spatial localization of a pMtIRE-gusA transgene in transformed roots of composite plants showed that MtIRE expression is confined to the proximal part of the invasion zone, zone II, found in indeterminate nodules. This suggests MtIRE is useful as an expression marker for this region of the invasion zone.