We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5' noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.