Gene transfection, a process used to study gene function, is itself poorly understood. Transfection-enhancing agents, including phorbol myristic acid (PMA) and histone deacetylase (HDAC) inhibitors, have been shown to increase transfection efficiency either by improving gene delivery into cells or by acting directly on delivered DNA sequences to increase their expression. Our results indicate that PMA and HDAC inhibitors can also regulate transfection efficiency by modulating distinct classes of cellular genes, which otherwise limit or block the expression of transfected genes already present in the nucleus. Either HDAC inhibitors or PMA was required to express reporter plasmids already present in the nucleus of lymphocyte lines. HDAC inhibitors and PMA seemed to operate through "transfection-controlling" cellular genes or gene products, rather than acting directly on transfected expression plasmids. PMA appeared to increase transfection efficiency by activating PKC-inducible, immediate-early gene products. Conversely, HDAC inhibitors functioned through a non-PKC-dependent pathway that required new protein synthesis, potentially acting through the de-repression of chromosomal genes. Neither delivery across the cell membrane nor into the nucleus may be rate-limiting for expressing transfected genes in some cell lines. In such cells, the targeted modulation of specific cellular genes may be required to efficiently express transfected genes.