Proenkephalin used to be thought of as a molecule destined exclusively for the secretory pathway of neurones and to be cleaved into small enkephalin peptides. We now know that uncleaved proenkephalin is a nuclear protein in a range of non-neural cell types including embryonic fibroblasts and mvoblasts where it responds to growth arrest and differentiation signals (Bouger and Spruce. 1995, JCB, 130, 1251-1262). The control of cell proliferation and differentiation is intimately coupled to the control of programmed cell death, a process in which proenkephalin is also involved and is governed by the localisation of proenkephaiin within the cell. Overexpression of transfected proenkephalin in the cytoplasm and secretory pathway of stable cell lines represses apoptosis induced by o.xidanls and staurosporine. Transfected proenkephalin can also be targeted to the cell nucleus either by forcing translation initiation from an alternate site or by removal of the signal peptide sequence. Cells in which the product of the tumour suppressor gene p53 is functionally inactive, such as Saos-2 and COS cells, tolerate nuclear overexpression of proenkephalin; in contrast, elevated levels of nuclear proenkephalin induces 3T3 and Rat-1 fibroblasts, which have wild-type p53 function, to die by apoptosis. Transfection of proenkephalin destined for the nucleus into cell lines containing a temperature sensitive mutant form of p53 (MCF-7::p53vall35 breast carcinoma cells, and Clone 6 :p53vall35 rodent fibroblasts) die by apoptosis within 24 to 48 hours at the permissive temperature (32 degC) but survive when p53 is inactivated; this has been shown in transiently and stably transfected cells and in cells microinjected with plasmid DNA encoding nuclear proenkephalin. In wild-type MCF-7 cells and in 3T3 and Ral-1 cells, which possess lower levels of functional p53, a lesser amount of death is induced by nuclear proenkephalin. Collectively, these data indicate that nuclear proenkephalin induces death in a p53 dose-dependent manner; experiments are underway to determine why this is so.