We report the characterization of vaccinia virus gene B12R which is predicted to encode a 33K protein with 36% amino acid identity to the serine/threonine protein kinase encoded by vaccinia virus gene B1R. S1 nuclease protection experiments showed that gene B12R is transcribed early during infection from an initiation site 11 bp upstream of the open reading frame (ORF). The gene encodes a 33K polypeptide that is not required for virus replication in tissue culture nor for virus virulence in a murine intranasal model. Expression of the B12R gene in Escherichia coli produced an abundant 33K polypeptide which lacked protein kinase activity under conditions in which the protein kinases encoded by vaccinia virus gene B1R and African swine fever virus gene j9L are active.