We have undertaken a detailed study to identify mechanisms regulating expression of NCRs. We used a custom Affymetrix oligonucleotide microarray to examine the expression changes of 566 NCRs in different stages of nodule development. Additionally, rhizobial mutants were used to understand the importance of the rhizobial components in induction of NCRs. Early NCRs were detected during the initial infection of rhizobia in nodules and continue to be expressed into the late stages of nodule development. Late NCRs were induced concomittant with bacteroid development in the nodules. The induction of these groups of genes was correlated with the number and morphology of rhizobia in the nodule. We used a custom Affymetrix chip containing 684 probe sequences of Medicago DEFLs to explore the expression patterns of NCRs in nodules inoculated with Sinorhizobium meliloti 1021(Sm1021) at marked developmental stages and nodules inoculated with various mutants derived from Sm1021 totalling 14 different treatments. Each treatment was supported by three biological replicates giving a grand total of 42 samples.