We have isolated from an established Aedes albopictus (mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, crossresistance to colchicine, and sensitivity to Tween-80 indicated that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistance in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.