Hyperpolarization-activated inward current (I(h)) has been shown to be involved in production of bursting during various forms of rhythmic activity. However, details of I(h) in spinal interneurons related to locomotion remain unknown. Using Cfos-EGFP transgenic mice (P6-P12) we are able to target the spinal interneurons activated by locomotion. Following a locomotor task, whole cell patch-clamp recordings were obtained from ventral EGFP+ neurons in spinal cord slices (T(13)-L(4), 200-250 microm). I(h) was found in 51% of EGFP+ neurons (n = 149) with almost even distribution in lamina VII (51%), VIII (47%), and X (55%). I(h) could be blocked by ZD7288 (10-20 microM) or cesium (1-1.5 mM) but was insensitive to barium (2-2.5 mM). I(h) activated at -80.1 +/- 9.2 mV with half-maximal activation -95.5 +/- 13.3 mV, activation rate 10.0 +/- 3.2 mV, time constant 745 +/- 501 ms, maximal conductance 1.0 +/- 0.7 nS, and reversal potential -34.3 +/- 3.6 mV. 5-HT (15-20 microM) and ACh (20-30 microM) produced variable effects on I(h). 5-HT increased I(h) in 43% of EGFP+ neurons (n = 37), decreased I(h) in 24%, and had no effect on I(h) in 33% of the neurons. ACh decreased I(h) in 67% of EGFP+ neurons (n = 18) with unchanged I(h) in 33% of the neurons. This study characterizes the I(h) in locomotor-related interneurons and is the first to demonstrate the variable effects of 5-HT and ACh on I(h) in rodent spinal interneurons. The finding of 5-HT and ACh-induced reduction of I(h) in EGFP+ neurons suggests a novel mechanism that the motor system could use to limit the participation of certain neurons in locomotion.