Several noncognate tRNA's from Escherichia coli were mischarged with glutamine by E. coli glutaminyl-tRNA synthetase if dimethylsulfoxide was present in the reaction mixture. Kinetic analysis of the mischarging revealed that dimethyl sulfoxide stimulated the misacylation by affecting the maximum velocity. Several noncognate tRNA's were shown to interact with glutaminyl-tRNA synthetase as measured by their ability to protect the enzyme against thermal inactivation or to replace cognate tRNA in stimulating glutamine-dependent ATP-PPi exchange reaction. These tRNA's, however, did not coincide with those which were mischargeable with glutamine.