Testicular peritubular cells are myofibroblastic cells, which represent the major cellular components of the wall of the seminiferous tubules. In men their phenotypic characteristics, including possible secretory activity and regulation, are not well known, either in normal or pathologically altered testes. Especially in testes of men with impaired spermatogenesis the cytoarchitecture of the tubular wall is frequently remodelled and presents fibrotic thickening, increased innervation, and infiltration by macrophages (MPs) and mast cells (MCs). The latter are both sources of TNFalpha. The purpose of our study was to explore human testicular peritubular cells and mechanisms of their regulation. To this end we primarily studied cultured human testicular peritubular cells (HTPCs), isolated from adult human testes. Having established that HTPCs express TNFalpha receptors 1 and 2 and respond to recombinant human TNFalpha by a rapid phosphorylation of ERK1/2, we used complementary approaches, including genearray/RT-PCR studies, Western blotting/immunocytochemistry, and ELISA techniques, to study phenotypic characteristics of HTPCs and actions of TNFalpha. We found that HTPCs express the NGF gene and TNFalpha stimulated mRNA levels and secretion of NGF in a dose- and time-dependent manner. Similarly, MCP-1 was identified as a product of HTPCs, which was regulated by TNFalpha in a concentration- and time-dependent way. TNFalpha furthermore strongly enhanced expression and/or synthesis of other inflammatory molecules, namely IL-6 and COX-2. Active COX-2 is indicated by increased prostaglandin D2 levels. In addition, ICAM-1, which was not detected at protein level in the absence of TNFalpha, was induced upon TNFalpha stimulation. In conclusion, these results provide novel insights into the nature of human peritubular cells, which are able to secrete potent signaling molecules and are regulated by TNFalpha. These results also hint to an as yet unknown role of peritubular cells in normal human testis and involvement in the patho-mechanisms associated with impaired spermatogenesis in men.