This study examined whether bacterial cell products that might gain access to the intestinal interstitium could activate mouse colonic nociceptive dorsal root ganglion (DRG) neurons using molecular and electrophysiological recording techniques. Colonic projecting neurons were identified by using the retrograde tracer fast blue and Toll-like receptor (TLR) 1, 2, 3, 4, 5, 6, 9, adapter proteins Md-1 and Md-2, and MYD88 mRNA expression was observed in laser-captured fast blue-labeled neurons. Ultrapure LPS 1 μg/ml phosphorylated p65 NF-κB subunits increased transcript for TNF-α and IL-1β and stimulated secretion of TNF-α from acutely dissociated DRG neurons. In current-clamp recordings from colonic DRG neurons, chronic incubation (24 h) of ultrapure LPS significantly increased neuronal excitability. In acute studies, 3-min superfusion of standard-grade LPS (3–30 μg/ml) reduced the rheobase by up to 40% and doubled action potential discharge rate. The LPS effects were not significantly different in TLR4 knockout mice compared with wild-type mice. In contrast to standard-grade LPS, acute application of ultrapure LPS did not increase neuronal excitability in whole cell recordings or afferent nerve recordings from colonic mesenteric nerves. However, acute application of bacterial lysate (Escherichia coli NLM28) increased action potential discharge over 60% compared with control medium. Moreover, lysate also activated afferent discharge from colonic mesenteric nerves, and this was significantly increased in chronic dextran sulfate sodium salt mice. These data demonstrate that bacterial cell products can directly activate colonic DRG neurons leading to production of inflammatory cytokines by neurons and increased excitability. Standard-grade LPS may also have actions independent of TLR signaling.