Cells with irregular elongated shape and numerous long thin processes were described earlier in guinea-pig mesenteric arteries (Pucovsky et al. 2003). Due to their morphological similarity with the Interstitial Cells of Cajal (ICCs), they were named the arterial ICC-like (AIL) cells. The ontogenic origin of these cells, suggestive of their physiological role, is as yet unknown. This work investigates the AIL cells for the presence of smooth muscle cell (SMC) and neural marker proteins in order to elucidate which lineage they belong to. Guinea-pigs were humanely killed. Single cells were obtained by enzymic digestion of mesenteric arteries. The cells were fixed, labelled by incubation with primary antibodies against a target molecule and the labelling was visualised using fluorescent secondary antibodies. The images were obtained with a laser scanning confocal microscope. Statistical significance was tested with Student’s t test. The staining for α-smooth muscle actin, an early marker of SMC lineage, was confined to the subplasmalemmal cytoplasm and the filopodia of the AIL cells (signal registered from 89.1±2.4% of pixels within 1 μm of plasma membrane and only from 15.4±6.5% of pixels more than 1 μm away from the plasma membrane, n=10, p=7×10^-7). Similar distribution was found in SMCs (78.8±4.5% of fluorescent pixels within 1 μm of plasma membrane and 4.3±2.0% more than 1 μm away, n=13, p=1.9×10^-9). Neither AIL cells nor SMCs stained for neuronal marker PGP 9.5. When pairs consisting of one SMC and one AIL cell were stained for smooth muscle myosin heavy chain, a marker of differentiated SMCs, the AIL cells produced a fluorescence signal of similar intensity to the SMCs (98.9±5.8% of the signal from the SMCs; n=7 pairs, p=0.852). Staining of the SMC/AIL cell pairs for smoothelin, a component of cytoskeleton and the most specific marker of contractile SMCs to date, showed significantly weaker signal in AIL cells (41.4±6.7% of the signal from the SMCs; n=10 pairs, p=10^-5). In all the experiments the primary or secondary antibodies alone produced none or negligible fluorescence. The data suggest that the AIL cells are closely related to contractile SMCs and are not fibroblasts, myofibroblasts or cells of neural origin.