1 The characteristics of transmembrane currents evoked by 5-hydroxytryptamine (5-HT) in the neuroblastoma x Chinese hamster brain cell line NCB-20 and neuroblastoma clonal cell line N1E-115 have been studied under voltage-clamp conditions by the whole-cell recording and outside-out membrane patch modes of the patch-clamp technique. 2 In 73% of NCB-20 cells examined (n = 221), and all N1E-115 cells studied (n = 80), 5-HT (10 microM) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5-HT reversed in sign at a potential of approximately -2 mV and demonstrated inward rectification. 3 The reversal potential of 5-HT-induced currents (E5-HT) recorded from either NCB-20 or N1E-115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E-115 cells, reducing internal K+ concentration from 140 to 20 mM produced a positive shift in E5-HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mM was associated with a negative shift in E5-HT of about 37 mV. A large reduction in internal Cl- concentration (from 144 to 6 mM) had little effect on E5-HT. 4 5-HT-induced currents of NCB-20 cells were unaffected by methysergide (1 microM) or ketanserin (1 microM), but were reversibly antagonized by GR38032F (0.1-1.0 nM) with an IC50 of 0.25 nM. GR 38032F (0.3 nM) reduced 5-HT-induced currents in N1E-115 cells to approximately 26% of their control value. 5 On outside-out membrane patches excised from both NCB-20 and N1E-115 cells, 5-HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nM) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5-HT (1 microM) in N1E-115 cells suggests that 5-HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside-out membrane patches to 5-HT cannot at present be resolved into clear single channel events.