Introduction: Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney. Methods: To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample. As proof of concept, we used our technique with transplant kidney biopsy samples to provide examples of clinically relevant immune information obtainable with cytometry. Results: A ratio of CD8+ to CD4+ lymphocytes greater than or equal to 1.2 in transplanted allografts is associated with rejection, even before it is apparent by microscopy. Elevated numbers of CD45 leukocytes and higher levels of interleukin (IL)−6, IL-8, and IL-10 indicate more severe injury. Antibody binding to renal microvascular endothelial cells can be measured and corresponds to antibody-mediated forms of allograft rejection. Eculizumab binding to endothelial cells suggests complement activation, which may be independent of bound antibody. We compared intrarenal leukocyte subsets and activation states to those of peripheral blood from the same donor at the time of biopsy and found significant differences; thus the need for new techniques to evaluate immune responses within the kidney. Conclusion: Assessment of leukocyte subsets, renal microvascular endothelial properties, and measurement of cytokines within a renal biopsy by flow cytometry enhance understanding of pathogenesis, indicate disease activity, and identify potential targets for therapy. Keywords: cytokines, cytometry, immunology, kidney biopsy, rejection