Covalent joining of the two half molecules of tRNAAla by T4 RNA ligase to form a reconstituted whole molecule was investigated. The two half molecules consisting, respectively, of residues 1-35 and 36-75 were prepared by partial degradation of tRNAAla with RNAase T1. The 5'-half molecule was treated with alkaline phosphatase to remove the 3'-terminal phosphate group, and the 5'-OH group of the 3'-half molecule was phosphorylated with [gamma-32P]ATP by polynucleotide kinase. The two terminal nucletides to be joined were identified as Guo and Cyd. Prior to the covalent joining reaction, the two modified half molecules in an equimolar mixture were annealed, and the rejoined half molecules, separated by gel electrophoresis, served as the substrate for T4 RNA ligase. Optimum conditions for this ligation, such as RNA ligase concentration, pH, Mg2+ concentration, reaction temperature and time of reaction, were investigated. Under the optimum conditions a yield of about 70% joining of the reconstituted whole molecule was obtained as shown by gel electrophoresis, resistance to hydrolysis by alkaline phosphatase, nearest neighbour analysis and alanine acdeptor activity.