155 Transplant allograft rejection is a complex phenomenon involving several different cell types and the intricate transcriptional programs of each of these cell types in concert. Although the basic sequence of HLA restricted recognition of antigen and subsequent activation of a generalized inflammatory response as well as a specific, primarily T cell directed, response is well characterized, we in fact have no real global understanding of this very complicated process. Genomic analysis techniques offer extremely powerful tools to decipher the natural history of rejection on a molecular level, there by leading to a better fundamental understanding of the phenomenon and to suggest potential targets for diagnostic, prognostic and the rapeutic purposes. We compare RNA from the peripheral blood mononuclear cells (PBMCs) of pediatric kidney transplant recipients undergoing episodes of biopsy proven acute rejection with PBMC RNA from pediatric kidney transplant recipients without evidence of rejection (postoperative day 7). One microgram of total RNA is purified from each patient, subjected to an RNA amplification procedure and then labeled with fluorescent nucleotides in a reverse transcriptase reaction (Cy-3 dUTP for nonrejecting and Cy-5 dUTP for rejecting samples). The two probe mixtures are combined and hybridized to cDNA microarrays containing 9,600 unique human gene targets. The microarrays are then scanned with a confocal laser microscope, allowing for quantitation of relative mRNA abundances between the two starting RNA populations for any given gene present on the array. In analyzing the first four such experiments, we find that genes previously known to be important in acute rejection are upregulated, including T cell receptor alpha (13.9 fold induction), NKG5/granulysin (13.4 fold), MHC class II DP beta (5.4 fold), CD8 alpha (3.4 fold) and IL-2R (3.0 fold). A prominent interferon response is noted as well. In addition, several ESTs representing genes of unknown function are upregulated more than two fold in the rejection samples. A large subset of these genes are consistently upregulated in all four experiments performed to date-and include NKG5/granulysin, T cell markers, several interferon inducible genes and ESTs. These results encourage us to pursue potential peripheral blood markers, both of known and unknown genes, that may be specific for acute rejection. Correlation with results obtained from allograft biopsy specimens and application of gene clustering bioinformatics algorithms to these large datasets will help focus the search for genes and transcriptional pathways central to rejection.