Rat hippocampal neurons in culture extended their neurites until day 5 in vitro (DIV). Then, the mean neuritic length slightly decreased. Excitatory amino acid (EAA)-elicited inositol phosphate (IP) formation increased from 0.5 to 2 DIV, reached a plateau between 2 and 4-5 DIV, and then gradually decreased until 10 DIV. This decrease was likely not due to neuronal death. This developmental pattern was observed for N-methyl-D-aspartate, kainate, glutamate, ibotenate and quisqualate (QA). Interestingly, the 1S,3R-aminocyclopentane dicarboxylate (1S,3R-ACPD) response slightly increased during neuronal culture development. At 3 DIV, the ionotropic antagonists 6,7-dinitro-quinoxalin-2,3-dion and D-2-amino-5-phosphonopentanoate efficiently blocked N-methyl-D-aspartate and kainate-elicited IP formation, and partially inhibited glutamate and ibotenate responses. QA and 1S,3R-ACPD responses were not affected, suggesting a metabotropic action for these two compounds. Furthermore, QA and 1S,3R-ACPD potencies significantly increased between 3 and 10 DIV. The transient high activity periods induced by EAA, except for 1S,3R-ACPD, are not observed for norepinephrine, carbachol and potassium chloride responses. Taken together, these data suggest that: (i) QA and 1S,3R-ACPD can act on two different glutamate metabotropic receptors subtypes during development; and (ii) the EAA-induced transient peaks of IP stimulation, which are specific with respect to other neuroactive substances profiles, could be involved in the development of hippocampal neurons. Indeed, these transient high activities take place when the neuritic length regularly increases in vitro.