We have developed an in vitro culture system which supports the differentiation of Drosophila pupal wings. Cultured wings develop marginal bristles and wing veins, and wing cells form a single prehair at their distal vertex at the appropriate developmental stages. We have tested two molecules with well defined activities to determine the usefulness of this system for applying pharmacological approaches to wing differentiation. Cycloheximide (CY) is a small molecule which inhibits protein synthesis. We found that 50 nM CY rapidly blocks all stages of wing differentiation without lowering cell viability. Chitinase is an enzyme which cleaves chitin polymers and is involved in normal cuticle apolysis. Chitinase applied prior to 28 h apf caused a contraction of the wing without affecting the general wing pattern. We have detected connections between the epithelium and pupal cuticle that are presumably targets of chitinase and are necessary for maintaining normal tissue shape during morphogenesis. Later in development exposure to chitinase caused a loss of normal prehair and bristle polarity, and high doses resulted in a severe disruption of the actin cytoskeleton.