Abstract Daily variations in the in vitro haemolymph juvenile hormone esterase activity (hJHE) of adult male and female Periplaneta americana were monitored over a 2 week period from the time of adult emergence and throughout the first reproductive cycle of the adult female. Kinetic analysis of hJHE from females indicated an apparent K m JH III of 5.59±1.75 μ m ( V max=140 pmol JH III hydrolysed h −1 per μl haemolymph). In females the mean rate of JH III metabolism in diluted haemolymph shortly after emergence was 27.5±1.5 pmol h −1 μl −1 ( n=16) and remained relatively low (16–32 pmol h −1 μl −1) over much of early adult development. Activity remained at this level during the first two days of the 4 day reproductive cycle, but showed a much increased broad peak of activity (74–93 pmol h −1 μl −1) at 60–72 h post-extrusion. This peak lags behind the whole body JH titre peak, which could suggest that elevated levels of JH III may bring about the induction of JH esterase(s). A different pattern of JHE activity was observed in adult males. hJHE rates in males at emergence were almost twice as high (81.5±15.8 pmol h −1 μl −1, n=16) as those found in females at this time, but thereafter showed a downturn to moderate levels (44–68 pmol h −1 μl −1) that were maintained for the remainder of the study. Rapid (FPLC) DEAE–sepharose ion exchange chromatography, ultrafiltration and fast-flow superose gel filtration chromatography were employed to achieve an efficient partial purification (166-fold) of the hJHE from cell-free plasma of reproductively active female P. americana 48–72 h post-ootheca extrusion. Gel filtration and SDS–polyacrylamide gel electrophoresis (PAGE) revealed an enzyme having apparent molecular mass of between 60 and 70 kDa, whilst non-denaturing PAGE and iso-electrofocusing resolved a single acidic enzyme peak with a pI of 4.9.