In the olfactory organ of the squid, Lolliguncula brevis there are five morphological types of olfactory receptor neurons (ORNs). Previous work to characterize odor sensitivity of squid ORNs was performed on only two of the five types in dissociated primary cell cultures. Here, we sought to establish the odorant responsiveness of all five types. We exposed live squid or intact olfactory organs to excitatory odors plus the activity marker, agmatine (AGB), an arginine derivative that enters cells through nonselective cation channels. An antibody against AGB was used to identify odorant-activated neurons. We were able to determine the ORN types of AGB-labeled cells based on their location in the epithelium, morphology and immunolabeling by a set of metabolites: arginine, aspartate, glutamate, glycine, and glutathione. Of 389 neurons identified from metabolite-labeled tissue, 3% were type 1, 32% type 2, 33% type 3, 15% type 4, and 17% type 5. Each ORN type had different odorant specificity with type 3 cells showing the highest percentages of odorant-stimulated AGB labeling. Type 1 cells were rare and none of the identified type 1 cells responded to the tested odorants, which included glutamate, alanine and AGB. Glutamate is a behaviorally attractive odorant and elicited AGB labeling in types 2 and 3. Glutamate-activated AGB labeling was significantly reduced in the presence of the adenylate cyclase inhibitor, SQ22536 (80 microM). These data suggest that the five ORN types differ in their relative abundance and odor responsiveness and that the adenylate cyclase pathway is involved in squid olfactory transduction.