We examined the effects of environmental regulation of gene transcription on the accuracy of a transcriptional profiling method for determining insect age. In combined temperature/nutrition treatments, Aedes aegypti (L.) mosquitoes were maintained in the laboratory at three different temperatures (20, 26, and 32 degrees C), and larvae were fed on low, medium, and high diet regimens. Adult mosquitoes of distinct size classes were produced. Transcription of three age-responsive genes (Ae-15848, Ae-8505, and Ae-4274) was measured from 1-, 10-, and 19-d-old specimens using a quantitative reverse-transcription polymerase chain reaction method incorporating dual-labeled TaqMan probes. Temperature had a significant effect on transcript abundance for two of the model genes (Ae-15848 and Ae-8505), and transcription of model genes was unaffected by the main effect of larval diet level; however, significant temperature by diet level interactions were observed. Total RNA yield from individual mosquitoes varied according to adult age and temperature, and when combined with wing length, provided a useful predictor variable in age prediction models. More accurate age predictions were achieved from models generated at the same temperature as test mosquitoes; however, whereas significant differences in mean predicted ages were observed between 1- and 10-d-old mosquitoes, differences between 10 and 19 d were nonsignificant. This study highlights the effect of environmental regulation on gene transcription age grading and the need to identify additional gene biomarkers of age to improve the classification of older mosquitoes.