Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5-HT1A-R), could cause attenuation of the activity of both N-type Ca2+ channels and N-methyl-D-aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5-HT1A-R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron-derived cell line (HN2-5) that is Ca2+ channel-deficient, we demonstrate here that an alternative pathway is responsible for 5-HT1A-R-mediated protection of these cells from anoxia-triggered apoptosis, assessed by deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL). The 5-HT1A-R agonist-evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation-mediated activation of mitogen-activated protein kinase (MAPK), as evidenced by the elimination of the agonist-elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5-HT1A-R caused a 60% inhibition of anoxia-stimulated caspase 3-like activity in the HN2-5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5-HT1A-R caused an activation of PI-3Kγ in HN2-5 cells, our results showed that this PI-3Kγ activity was not linked to the 5-HT1A-R-promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2-5 cells, agonist binding to the 5-HT1A-R results in MAPK-mediated inhibition of a caspase 3-like enzyme and a 60-70% suppression of anoxia-induced apoptosis through a Ca2+ channel-independent pathway.