A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens transparency. In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing 15 micrograms/ml insulin plus 1-2 ng/ml BB platelet-derived growth factor (PDGF), or 5-7 ng/ml epidermal growth factor (EGF) allowed similar growth in vitro, during the same time period. Normal lens growth occurred in culture when fresh medium was delivered to lenses as a pulse every 4-6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF stimulates lens growth during development in vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation, differentiation, and receptor regulation in a developing tissue.