Cryosurgery, the use of low temperatures to destroy unwanted tissue, has emerged as a viable option in the treatment of cancerous lesions. Critical to the continual improvement of treatment efficacy is deciphering the biochemical responses of cells to low temperature exposure. To that end, the identification of delayed onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptotic pathways. We have hypothesized that in addition to delayed apoptotic events that have been shown to occur following exposure to mild sub-freezing temperatures (10 to −25°C), cells exposed to ultra-low temperatures (<−30°C) may also undergo a more rapid, early onset apoptosis.Human prostate cancer cells (PC3) were exposed to temperatures of −60, −30, and −15°C to simulate a cryoablative procedure. Using a combination of flow cytometry, fluorescent microscopy, and western blot analyses, samples were assessed at various times post-thaw to identify the presence, levels, and pathways involved in cell death. The results indicate that at ultralow temperatures (below −30°C) a significant population of cells undergoes programmed cell death within as little as 30min of thawing, attaining a maximum of ∼40% at 90min, and by 6h post-thaw only necrosis was observed. At elevated sub-freezing temperatures (above −30°C), however, the activation and progression of apoptosis occurs in a more delayed manner and is not noted until 6–24h post-thaw. The levels of apoptosis are also significantly lower with ∼10% of cells undergoing apoptosis at 6h post-thaw following exposure −15°C and ∼25% at 6h post-thaw for cells frozen to −30°C. Additionally, it was found that early onset apoptosis progressed through a membrane mediated caspase dependent mechanism.These data demonstrate the impact of apoptotic continuum whereby the more severe the cryogenic stress activates the extrinsic, membrane regulated apoptotic pathway while less severe freezing activates the intrinsic, mitochondrial mediated path. The rapid induction and progression of apoptosis at ultralow temperatures provides an explanation as to why such results have not previously been identified following freezing both in cryosurgical or cryopreservation settings. Ultimately, it is our aim to decipher the events and signaling pathways that are specifically involved in triggering rapid-onset apoptosis. Once known, cryosurgical procedures might be modified such that rapid-onset and delayed programmed cell death pathways can be selectively induced, in an effort to improve the overall efficacy of cryoablation.