The 60 pS channel, which mainly carries muscarinic receptor-gated cation current (mI_CAT) in ileal myocytes, could rarely be activated by carbachol in cell-attached patches (4 out of 77) although the channel activity more readily appears after patch excision with carbachol in the bath (19 out of 62). It thus appears that some native intracellular factors inhibit mI_CAT of which polyamines (PAs), the ubiquitous aliphatic polycations known to block various ion channels, seem the likely candidates. The effects of extra- and intracellular PAs (spermine and putrescine) on the mI_CAT evoked either by external carbachol or by internal GTP_γS were studied using patch-clamp techniques. Experiments were performed on single collagenase-dispersed myocytes isolated from the small intestine of humanely killed adult guinea-pigs. External PAs rapidly and reversibly inhibited mI_CAT in a concentration- and voltage-dependent manner. Cation conductance activation curve was N-shaped both before and after PA application although membrane depolarization significantly relieved the inhibition. The concentration producing 50% current inhibition at -40 mV was 1.03 (n=16) and 4.17 mM (n=14) for spermine and putrescine, respectively. The mI_CAT values were similar for both carbachol- and GTP_γS-evoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the PAs action. External spermine and putrescine reversibly inhibited the activity of the 60 pS muscarinic cation channel by reducing both unitary conductance and open probability. In the perforated-patch configuration (0.1 mg ml^-1 amphotericin B in the pipette) mInullCATnull in response to 100 μM carbachol was reduced on average by 65% compared to the conventional whole-cell recordings. However, in the latter case adding spermine to the pipette solution at 0.3 μM (which corresponds to its physiological cytoplasmic concentration) inhibited mI_CAT by 39±3% (n=19) without any noticeable change of the conductance curve shape. Internal putrescine was less effective, inhibiting mI_CAT by 31±3% at 10 mM (n=18). It is concluded that endogenous PAs, spermine in particular, strongly inhibit mI_CAT and this effect can contribute to their well-known suppressing effect on the gastrointestinal tract excitability and contractility.