The DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17 + in the region coding for the polypeptide ICP34.5 predicts a protein of 248 amino acids with a proposed M r of 26158. The entire RL1 open reading frame was cloned into the expression vector pET8c to enable over-expression of ICP34.5 in Escherichia coli. The expressed protein was partially purified and used as an immunogen to produce a polyclonal antiserum in rabbits. Construction of an ICP34.5 null mutant (1771), demonstrated that the predicted open reading frame for ICP34.5 in strain 17 + is correct and confirmed that HSV-1 strain 17 + ICP34.5 specifically determines neurovirulence. The specificity of the antiserum directed against the E. coli-expressed ICP34.5 was defined by Western blotting of wild-type and RL1-negative infected cell extracts.