Two cell lines isolated from Rhipicephalus appendiculatus (RAE 25) and Anocentor (= Dermacentor) nitens (ANE 58) responded to the intervertebrate hormones 20-hydroxyecdysone (20-HE) and juvenile hormone III (JH III) in vitro. In the presence of 0.2 or 2 nMolar 20-HE, the cells of the continuous line RAE 25 attached to the culture substrate at a rate of 9% pr hr for the first 8 hr, as did cells in growth medium. Twenty or 200 nMolar of 20-HE reduced the rate of cell attachment to 6% per hr, and in the higher hormone concentration the cells ceased to attach after 4 hr. Low concentrations (0.2 and 2 nMolar) of 20-HE stimulated the growth of the RAE 25 line (P < 0.02), but 200 nMolar or more inhibited growth (P < 0.001). Twenty-HE suppressed the growth of the young line ANE 58 in a dose-dependent manner, but the decrease in cell growth was less pronounced than in RAE 25. Ten to 100 times more (2 and 20 μMolar) 20-HE was needed to achieve significant growth suppression (P < 0.025 and < 0.005). The growth of both lines declined (P < 0.01) by 20% (RAE 25) or 30% (ANE 58) when the medium contained 38 μMolar of JH III. The bimodal growth response of line RAE 25 to 20-HE also occurred in the presence of 3.8 and 38 μMolar JH III, and 2 nMolar 20-HE counteracted the suppressive effect of 38 μMolar JHIII. The bimodal growth response of line RAE 25 to 20-HE also occurred in the presence of 3.8 and 38 μMolar JH III, and 2 nMolar 20-HE counteracted the suppressive effect of 38 μMolar JH III. Under the influence of 20-HE, the epithelial-like RAE 25 cells aggregated, detached and formed vesicles after certain times, depending on hormone concentration: 5 to 6 days with 2 nMolar, 2 days with 20 nMolar, and 1 day with 200 nMolar or more. The hemocytelike cells of ANE 58 were less sensitive to 20-HE. In the presence of 2 to 20 μMolar 20-HE, the pseudopodia retracted and 70% of the cell population detached. The responses of acarine cells to the hormones 20-HE and JH III are contrasted with those reported for insect cells in culture.