The mammalian carotid body consists of preneural type I (glomus) cells synaptically coupled to afferent axon terminals and enveloped by type II (sustentacular) cells. Recent studies indicate the presence of multiple putative neurotransmitters in this arterial chemoreceptor organ. A double-labeling immunocytochemical technique was utilized which allows simultaneous visualization of two neurochemicals in a single cell. The issue of transmitter co-occurrence in type I cells of the cat carotid body was addressed using specific antibodies for seven neurochemical agents: tyrosine hydroxylase, dopamine-beta-hydroxylase, choline acetyltransferase, serotonin, substance P, met-enkephalin and chromogranin. A high degree (greater than 70%) of co-localization was observed for most pairs of markers, indicating the co-existence of multiple neuroactive agents in type I cells of the cat carotid body. The intensity of staining varied greatly among cells but formed a pattern. Thus, for tyrosine hydroxylase and dopamine-beta-hydroxylase, the majority of double-labeled type I cells exhibited equivalently low or high levels of both, while for the neuropeptides unequal levels of the two markers predominated. Neuropeptides also co-existed in type I cells with catecholamine-synthesizing enzymes and with serotonin. The functional significance of such patterns of multiple co-existence involving biogenic amines and neuropeptides is discussed. Our results indicate a high degree of co-occurrence of reaction product for amine-synthesizing enzymes (tyrosine hydroxylase, dopamine-beta-hydroxylase and choline acetyltransferase), the indoleamine serotonin, and the neuropeptides substance P and met-enkephalin.