Muscarinic acetylcholine receptors regulate distal airway resistance and secretion. The subtype expressed in the lung in different species remains uncertain. It has recently become possible to identify the M4 subtype by careful comparison of antagonist affinities. We characterized the binding of [3H]quinuclidinyl benzilate ([3H]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2-8 week old pigs in comparison to cloned human M3 and M4 receptors expressed in COS cells, to M2 in rat atria and to M4 in bovine adrenal medulla. In porcine lung, [3H]QNB bound with high affinity (Kd = 95 +/- 9 pM) to a single homogeneous population of muscarinic receptor sites (Bmax = 340 +/- 10 fmol/mg protein). Competition studies showed that the affinity (expressed as pKi) of 3 selective blockers was in close agreement between pig lung and cloned human m4 (r = 0.996). A series of 10 blockers showed affinities closely matching reported values for M4 receptors of the adrenal medulla (r = 0.965). Conversely, affinity values in porcine lung differed significantly (P < 0.05, t-test) from those determined in parallel with either human cloned M3 or with rat atria expressing the M2 subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M4 subtype, in contrast to the M3 subtype typical of large airways in this species.