Three organophosphate resistant Boophilus microplus Canestrini cell lines were generated by exposing B. microplus VIII-SCC cell line to incrementally increased toxic concentrations of the acaricide coumaphos. The development of resistance was evidenced by LC50 values elevated over those of control cells. The resistant cell lines selected in higher concentrations of organophosphate, designated C44 and C54, also had significantly slower duplication rates than a resistant cell line selected in lower concentrations of coumaphos (C34) and the nonresistant control cells. Resistant cell lines C44 and C54 also had significantly higher levels of esterase after exposure to coumaphos than resistant cell line C34 and the nonresistant controls. These in vitro results agree with reports of increased esterase activity associated with organophosphate resistance in B. microplus ticks in vivo.