The plasma membrane calcium ATPase (PMCA) is a pump that extrudes calcium from the cytosol. It has four major isoforms of which 1 and 4 are ubiquitously expressed. In recent years, it has been reported that PMCA4 is involved in apoptosis via regulation of intracellular calcium levels [Ca2+]i. However, it is not clear whether PMCA4 promotes or prevents apoptosis. Since PMCA4 has been reported to modulate the activity of neuronal nitric oxide synthase (nNOS) via physical interaction; and because nitric oxide (NO) has both pro and antiapoptotic potentials, it is possible that the PMCA4-nNOS interaction is involved in PMCA4-mediated modulation of apoptosis. To study the role of PMCA4 in apoptotic signalling, the effect of PMCA4 deletion on tumour necrosis factor α (TNFα)-induced apoptosis was examined and the relevance of the PMCA4-nNOS interaction was assessed. Mouse embryonic fibroblasts (MEF) were derived from PMCA4 knockout (PMCA4-/-) and wild-type (PMCA4+/+) embryos. Apoptosis was induced in MEF by TNFα and quantified by measuring the activity of the executioner caspases 3 and 7 and by flow cytometric analysis of annexin V/propidium iodide labelled cells. Activation of signal mediators in TNFα signalling pathways was assessed by measuring the activated (phosphorylated) forms relative to total expression by immunoblotting. [Ca]i was measured by fluorometry using the Ca2+ fluorophore fluo-3. In non-stimulated cells, growth rates, [Ca2+]i and apoptosis levels were similar in PMCA4+/+ and -/- MEFs. Following stimulation with TNFα (10ng/ml) for 24 hours, PMCA4-/- MEF had significantly lower (by 46%) caspase 3/7 activation compared to PMCA4+/+ MEF (p<0.01, n=14). This was confirmed by flow cytometry. In pursuing the mechanism underlying this phenotype, the activation patterns of TNFα signal mediators were assessed. The proapoptotic caspase-8, the antiapoptotic inhibitor of kappa kinase and extracellular regulated kinase1/2, as well as c-Jun N-terminal kinase which has pro- and antiapoptotic potentials were similarly activated in PMCA4/+/+ and -/- MEF. Interestingly, [Ca2+]i did not change in either group up to 4 hours after TNFα treatment. To investigate whether modulation of nNOS by PMCA4 was responsible for this phenotype, the effects of the nNOS inhibitor S-methyl thiocitrulline (SMTC) and the NO donor S-nitroso-acetyl penicillamine (SNAP)were assessed. Treatment with SMTC or SNAP prior to induction of apoptosis by TNFα eliminated differences in apoptosis levels between PMCA4+/+ and -/- MEF, either by decreasing apoptosis in the PMCA4+/+ MEF (SMTC effect) or increasing it in the PMCA4-/- MEF(SNAP effect). In conclusion, this work has shown that PMCA4 mediates susceptibility to TNFα-induced apoptosis, likely through modulation of nNOS signalling.