Replication-deficient adenoviruses expressing human interferon-alpha2b (HuIFN-alpha2b) or the hybrid IFN-alpha2alpha1 or those with the secretory signal deleted, whose express is driven by the alpha-fetoprotein (AFP) promoter, were constructed and characterized. Synthesis of IFN protein and secretion or intracellular retention were tested by Western blotting and immunoassay. Expression of IFN by the recombinant adenoviruses was restricted to cells that constitutively express AFP. In these cells, expression of both secreted and nonsecreted recombinant IFN resulted in inhibition of cell proliferation, resistance to viral infection, induction of major histocompatibility complex (MHC) class I expression, increased apoptosis, and activation of an IFN-stimulated response element (ISRE)-containing promoter. Also, the induction of protein kinase R (PKR), increased phosphorylation of Stat1, and accumulation of hypophosphorylated pRb were observed for both the secreted and nonsecreted IFN, suggesting that the nonsecreted IFN may act through a similar pathway. Hep3B cells, an AFP-positive line derived from a patient with hepatocellular carcinoma (HCC), were injected subcutaneously (s.c.) into athymic nude mice to generate established tumors. Intratumoral injection of recombinant adenoviruses expressing secreted as well as the nonsecreted IFN caused suppression of tumor growth. As the AFP promoter is activated in many HCC cells but is silent in normal cells, these constructs may be useful in restricting IFN effects to the tumor cells while reducing toxicity to the neighboring tissues.