Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.