We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.