The expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) in spinal ventral horn neurons was studied in the guinea pig after right knee joint immobilisation (RKJI). At 1 wk after RKJI, neurons in the ipsilateral ventral horn from L4 to S1 segments showed a moderate reactivity for NADPH-d staining. At 2 wk, NADPH-d labelled neurons were also observed in the contralateral ventral horn. Ipsilateral NOS immunoreactive cells were not detectable until wk 2. The intensity of NADPH-d and NOS labelled neurons in the bilateral ventral horns was sustained, peaking at the 4th wk after RKJI. In guinea pigs subjected to 4 wk of RKJI and subsequently released from the immobilisation for 2 and 4 wk, NADPH-d and NOS reactivity in ventral horn neurons diminished. The expression of NADPH-d positive neurons differed from that of NOS labelled neurons in terms of time interval, cell number and staining intensity, the latter being later, fewer and weaker. It is suggested that the induction and upregulation of NADPH-d and NOS are attributable to reduced activity of muscles acting on the knee joint after RKJI; the changes are reversible. It is speculated that increased levels of NO production are involved in protective mechanisms against possible neuronal degeneration as a consequence of target dysfunction.