To obtain direct evidence for the attachment of 5SrRNA-ribosomal L 5 protein particles (5 SRNP) and methionine-tRNA (tRNA^met) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNA^met complex was dissociated from the macromolecular ARS complex fraction by n-octyl-β-D-glucoside (Method I) or by ω-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNASepharose chromatography using partially purified tRNAmet in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final Superdex^TM200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNA^met complex (M_r 200, 000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L 5 to the microconcentrators which was used to concentrate the final product, a distinct L 5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5 SrRNA. Dot blot analysis using an antibody against ribosomal protein L 5 showed that L 5 was present in the Superdex^TM200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNA^met exists stably in the MetRS complex. 5 SRNP and 5 SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5 SRNP(A) plays a role as a positive effector of MetRS.