Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.