The gypsy moth enters diapause in late embryonic development as a fully differentiated pharate larva surrounded by an abundant supply of yolk. Although diapause is known to be regulated by the endocrine system in other insect species that have been intensively studied, the mechanism involved in regulation of diapause in the gypsy moth is unknown. The present study is part of a series designed to develop a fundamental understanding of the physiology of diapause in this species and to develop methods to manipulate it in the laboratory. In nature and in the laboratory, diapause is gradually terminated by prolonged chilling of the eggs for several months. Recently it was discovered that KK-42, a novel insect growth regulator with anti-hormonal properties, was effective in averting diapause in the gypsy moth. Results of the present study show that 14 day old eggs are highly sensitive to KK-42 and treatment at this stage prevents diapause in 85% of the test populations. Studies in which successive age groups, incubated at 25 °C then transferred to 5 °C, showed that eggs must be at least 12–13 days old to survive prolonged chilling and to hatch successfully after re-incubation at higher temperature. Eggs chilled at 14–15 days post-oviposition retained complete sensitivity to KK-42, whereas, 21–22 and 29–30 day old eggs were not responsive unless they were chilled for 4–6 weeks. These findings are important in developing methods for manipulating diapause in stockpiled eggs of the gypsy moth for use in biocontrol programs and also provides knowledge useful for further inquiry into the mechanism of diapause regulation in this species.