tRNA and an enzyme fraction which contain aminoacyl‐tRNA‐synthetases and tanformylase, were prepared from Mycoplasma laidlawii A. After chromatography of tRNA on benzoylated‐DEAE‐cellulose, amino acid acceptor activites were determined. One or more species of tRNA were found for the following amino acid (number of isoaccepting species in brackets): alanine (1), arginine (2), cysteine (1), glycine (1), isoleucine (2), leucine (3), methionine (2), phenylalanine (1), serine (3), tyrosine (1), and valine (3). The mthionine specific tRNA was separated into a homologously formylatable and a non‐formylatable species.Cross‐reactions in heterologous systems from M. laidlawii A, Escherichia coli, and yeast were studied in some detail for methionine and phenylalanine. Aminoacylation was possible in all combinations of methionine specific tRNAs and enzymes. Transformylase of mycoplasma formylates tRNA from E. coli, but none of the methionine specific tRNAs from yeast.The content of minor nucleotides in tRNA from M. laidlawii A was determined mainly from [32P]tRNA by two‐dimensional thin layer chromatography.The findings are compared with those reported for other mycoplasma species and discussed in view of the genetic relationship of mycoplasma other microorganisms.