The cytokine tumour necrosis factor-alpha (TNF) has been implicated in autoimmune diseases and may play an indirect role in activation of pain pathways. In this study we have investigated the possibility that TNF directly activates cultured neonatal rat dorsal root ganglion (DRG) neurones and provides a signalling pathway from cells in the immune system such as macrophages to sensory neurones. Expression of TNF receptor subtypes (TNFR1 and TNFR2) on sensory neurones was identified using immunohistochemistry, fluorescence-activated cell sorting analysis and RT-PCR. Biochemical and immunocytochemical analysis showed that TNF activated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) but not p42/p44 MAPK. TNF treatment evoked transient Ca2+-dependent inward currents in 70% of DRG neurones. These TNF-evoked currents were significantly attenuated by ryanodine or thapsigargin or by inclusion of BAPTA in the patch pipette solution. Responses were also evoked in subpopulations of cultured DRG neurones by human mutant TNFs that cross-reacted with rat receptors and selectively activated TNFR1 or TNFR2 subtypes. TNF-evoked transient increases in [Ca2+]i were also detected in 34% of fura-2-loaded DRG neurones. The link between TNF receptor activation and Ca2+ release from stores remains to be elucidated. However, responses to TNF were mimicked by sphingolipids, including sphingosine-1-phosphate, which evoked a transient rises in [Ca2+]i in a pertussis toxin-insensitive manner in fura-2-loaded DRG neurones. We conclude that distinct receptors TNFR1 and TNFR2 are expressed on cultured DRG neurones and that they are functionally linked to intracellular Ca2+ mobilisation, a response that may involve sphingolipid signalling.