Pineal glands were obtained from two young female black rhinoceri that had died as a result of postcapture trauma during a translocation exercise. Hydroxyindole‐O‐methyltransferase (HIOMT) from these pineal glands showed a peak activity at pH 8.2, although high activity extended over a fairly wide pH range (7.8–8.4). N‐acetylserotonin was the best hydroxyindolic substrate for the enzyme, although other hydroxyindoles were methylated, the relative affinities being similar to values previously reported for bovine HIOMT. Kinetic analyses revealed that black rhinoceros HIOMT was subject to substrate inhibition by both substrates at high concentration; this observation is unlikely to have physiological significance. The catalytic mechanism was found to be ordered Bi‐Bi, in which S‐adenosylmethionine is the obligatory first substrate to bind to the enzyme, such binding allowing for binding of the hydroxyindolic substrate followed by catalysis, products again leaving the catalytic site in a sequent