The ecdysteroid (ES) content of the soft tick Ornithodoros moubata was investigated during the five successive nymphal molting cycles by means of an enzyme immunoassay (EIA). Samples were submitted to esterase hydrolysis in order to release free ecdysteroids from the acyl-ester conjugates (AP = apolar products). Crude and hydrolysed extracts were then analyzed by EIA using two different antibodies, a monoclonal raised against 20-hydroxyecdysone (20E) and a polyclonal raised against ecdysone (E). With the crude extracts, each molting cycle was associated with an ES peak, occurring in the middle of the instar. 20E was preponderant during the first 2 nymph cycles, but the proportion of E and other ES increased during the following instars. The shape of the peak also seemed to be different. Hydrolysis with esterase revealed that the total immunoreactivity was increased by 10-to 20-fold as compared to crude extracts in N1, N2, and N3, then by approximately 50-and 80-fold in N4 and N5 (last instar), respectively. The use of both antibodies and of HPLC fractionation showed that not only 20E was conjugated as AP, but also two other ES tentatively identified as ecdysone and 2-deoxyecdysone. It is suspected that a conversion of conjugated 20E to less immunoreactive ES occurs. Arch.