DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP‐ribose) polymerase‐1 (PARP‐1) cell death pathway. PARP‐1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)⁺ to form poly(ADP‐ribose) on acceptor proteins. The mechanisms by which PARP‐1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD⁺ depletion by PARP‐1 and the role of NAD⁺ depletion in this cell death program. PARP‐1 activation was induced by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP‐1 activation led to a rapid but incomplete depletion of astrocyte NAD⁺, a near‐complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD⁺ restored glycolytic function and prevented cell death. The addition of non‐glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP‐1 activation. These studies suggest PARP‐1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD⁺ pool. Depletion of the cytosolic NAD⁺ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood–brain barrier. © 2004 Wiley‐Liss, Inc.