Critical assessment of intestinal immune responses requires the ability to characterize leukocytes from different anatomic locations as leukocytes from inductive sites such as Peyer's patches and lymphoid follicles vary significantly from their effector counterparts, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). This study describes (1) methods developed to isolate specific intestinal leukocyte populations with high yield and purity, (2) difficulties encountered in establishing a panel of monoclonal antibodies to assess phenotype, and (3) the phenotypic characterization of effector and inductive sites in the feline small intestine. We found that the phenotypic distribution of feline intestinal leukocytes was similar to that found in other species such as humans, macaques and mice. The majority of IEL were CD5(+) T-cells with less than 7% B-cells. CD8(+) T-cells comprised approximately 60% of the IEL with roughly half displaying CD8alphaalpha homodimers. Approximately 10% of IEL were CD4(+) T-cells. In the LPL, CD4(+) T-cells predominated at 42%, with 33% CD8(+) T-cells and 10% B-cells. As would be expected, B-cells predominated in Peyer's patches with 40% B-cells, 28% CD4(+) T-cells and 20% CD8(+) T-cells. Increased MHCII expression was found in the Peyer's patches as compared to the IEL and LPL. B7.1 expression was significantly higher in mucosal leukocyte populations as compared to organized lymphoid tissue in the periphery with expression detected on 65% of IEL and 53% of LPL. Plasma cells were found in all regions of small intestine examined with greater numbers in lamina propria and Peyer's patches. Lymphoblasts were only identified in inductive tissue. In general, no differences were found between the phenotype of mucosal leukocyte populations from specific pathogen free or random source cats. However, the percentage of CD4(+) CD25(+) T-cells was significantly greater in both IEL and LPL from random source animals. This study provides techniques and a baseline from which future studies of the feline intestinal immune system can be conducted.