1. The possible role of nicotinamide-adenine dinucleotide (NAD+) and cyclic adenosine diphosphate ribose (cADPR) as regulators of M-type K+ currents (IK(M)) has been studied in whole-cell patch-clamped NG108-15 mouse neuroblastoma x rat glioma cells that had been transformed to express m1 muscarinic acetylcholine receptors (mAChRs). 2. Pre-incubation of NG108-15 cells for 6-8 h with streptozotocin (2-5 mM) reduced NAD+ levels by 40-50%. Nicotinamide (2-5 mM) increased NAD+ levels and prevented depletion by streptozotocin. 3. Streptozotocin pretreatment reduced the inhibition of IK(M) produced by 100 microM acetylcholine (ACh) from 51.6 +/- 7.0 to 29.1 +/- 7.5%. This was prevented by simultaneous pre-incubation with 2 mM nicotinamide or by adding 2 mM NAD+ to the pipette solution. Neither procedure significantly affected the initial amplitude of IK(M). 4. Inclusion of 2 microM cADPR in the pipette solution induced a slow loss of IK(M) with a time constant of about 20 min. 5. It is concluded that mAChR-induced inhibition of IK(M) requires intracellular NAD+. This might be needed for the formation of cADPR as a regulator or messenger for IK(M) inhibition.