The replication defect in the temperature-sensitive mutant A1 of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus was confirmed by the absence of intracellular nucleocapsids in infected cells incubated at the restrictive temperature. After preamplification, the relative yield of the A1 N protein accumulated intracellularly after 1 h of incubation at the restrictive temperature was decreased by 50% that of the wild-type or revertant A1 N protein. This difference was not as apparent in pulse-chase experiments. The functional lesion in A1 was correlated with a structural alteration in the N protein on the basis of the thermolability of the template activity of the A1 N protein-RNA complex in in vitro transcription reactions and the covariance of this phenotype with the temperature-sensitive phenotype in a spontaneous A1 revertant. This correlation was consistent with a direct role of the N protein in replication and allowed the assignment of the N gene to complementation group A.