Abstract Prolyl- tRNA synthetase from plants (e.g. Delonix regia) containing azetidine-2-carboxylic acid (A2C), activated imino acid analogues larger than proline (Pro) more efficiently than did the enzyme from plants lacking A2C. The reverse situation was observed for analogues, including A2C itself, that are smaller than Pro. The enzyme from A2C-producing species was quite labile and salt-sensitive, with a high pH optima for the ATP- 32PPi exchange reaction, whereas the enzyme from non-producer species was stable and insensitive to salts, with a lower pH optimum. Certain analogues of Pro, which failed to stimulate ATP- 32PPi in the presence of a particular type of Pro- tRNA synthetase, nevertheless could bind to the enzyme and inhibit the esterification of tRNA by Pro. In the absence of tRNA, no significant ATP- 32PPi exchange was catalyzed by the Delonix enzyme on addition of A2C; the addition of tRNA resulted in a low but real level of activation of the analogue relative to Pro. These findings are discussed in relation to the ability of the enzyme from A2C-producing plants to discriminate against the analogue.