The ring gland (RG) of D. melanogaster is the major site of production and release of developmental hormones. Therefore, transgenic lines driving Gal4 in this specific tissue constitute a useful tool to investigate molecular pathways involved in the regulation of developmental processes. To obtain RG-Gal4 driver lines, we have transformed Drosophila with a pPTGAL vector containing a 67 bp fragment (-253/-187) from DNA puff C4 gene, which has been shown to drive transcription in the RG, starting in late embryos and continuing throughout development. Obtaining these transgenic lines was only possible when the transformed animals were kept at 18⁰C, instead of 25⁰C as routinely used, which suggests that the level of Gal4 driven by the pPTGal4-RG affects development. Since it is well known that the Gal4 activity levels in Drosophila are related to temperature, we have investigated the viability of pPTGAL-RG flies maintained at different temperatures to understand the effect of Gal4 expression in the ring gland. Two series of experiments were performed. In one, we compared the percentage of transgenic flies which emerged from animals maintained at 18⁰C with the percentage of emerged flies when the culture was transferred to 25⁰C, at different stages of development (embryo, L1, L2, L3, pupa).  In the second series of experiments, we verified the percentage of transgenic flies which emerged from animals transferred from 25⁰C to 18⁰C at the same developmental stages. The results show that in the cultures transferred from 18⁰C to 25⁰C, transgenic flies emerged only when the transference was done in L3 or pupal stage. When the culture was transferred from 25⁰C to 18⁰C, transgenic flies emerged when the temperature shift was performed either in the embryonic stage, or in L1 or in L2, but not after that. These results indicate that the activity of Gal4 in the RG affects development, particularly by interfering with the transition from the second to the third larval instar. These data, together with previous observations that Gal4 directed by the GMR promoter causes developmental defects and increased apoptosis in the eye, reinforces the notion that Gal4 can be active in Drosophila despite the fact that UAS promoter sequences are not found in the genome of this animal.