ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs(+) have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1-10 microM) inhibited a current that reversed at -43.7+/-3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K(+) (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I(h). Depolarisation-activated transient (I(K(A))) and sustained outward currents were unaffected by ZD-7288. The IC(50) for block of I(h) by ZD-7288 was around 0.2 microM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba(2+) and Rb(+) blocked an inwardly rectifying potassium (K(+)) current, confirming both the presence of I(K(IR)) and its insensitivity to ZD-7288. Cs(+) rapidly and reversibly blocked both I(h) and I(K(IR)). Inhibition of I(h) by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I(h), and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I(h) that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.