To determine if nitric oxide (NO) and Fos immunoreactivity induced by noxious stimulation were colocalized in spinothalamic neurons, double-staining immunocytochemical techniques were combined with retrograde neuroanatomical tracing procedures. Initial studies on three rats demonstrated that Fos and nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, did not coexist in spinothalamic tract neurons. However, some spinothalamic neurons were found to contain NOS and some NOS immunoreactive processes were found to appose Fos containing neurons. Thus the remainder of the study: (1) analyzed the relationship of NOS positive neuronal processes with Fos stained neurons using a Fos immunocytochemical technique in combination with either NOS immunofluorescence or NADPH-diaphorase histochemistry; and (2) quantitated the number of NOS containing cells that project to the thalamus using a combined immunofluorescent-retrograde tracing procedure. Both NOS-like immunoreactive (NOS IR) neuronal processes and NADPH-diaphorase positive neuronal processes in the dorsal horn of the lumbar spinal cord were found to appose Fos positive neurons located in laminae I and II of the dorsal horn. Approximately 40% of Fos-labeled cells in these superficial laminae were found to be in apposition to or in close proximity to NOS labeled neuronal processes. Examination of spinal cord sections for NOS-containing spinothalamic tract neurons revealed that lamina X was the only spinal cord region containing such double-labeled neurons. Further quantification revealed that approximately 10% of NOS positive neurons in lamina X were double-labeled with Fluorogold. These findings support the hypothesis that nitric oxide is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus and further indicate that nitric oxide may contribute to the central transmission of spinothalamic information.