Gene expression responds to the environment and can also evolve rapidly in response to altered selection regimes. Little is known, however, about the extent to which evolutionary adaptation to a particular type of stress involves changes in the within-generation (‘plastic’) responses of gene expression to the stress. We used microarrays to quantify gene expression plasticity in response to ethanol in laboratory populations of Drosophila melanogaster differing in their history of ethanol exposure. Two populations (‘R’ populations) were maintained on regular medium, two (‘E’) were maintained on medium supplemented with ethanol, and two (‘M’) were maintained in a mixed regime in which half of the population was reared on one medium type, and half on the other, each generation. After more than 300 generations, embryos from each population were collected and exposed to either ethanol or water as a control, and RNA was extracted from the larvae shortly after hatching. Nearly 2000 transcripts showed significant within-generation responses to ethanol exposure. Evolutionary history also affected gene expression: the E and M populations were largely indistinguishable in expression, but differed significantly in expression from the R populations for over 100 transcripts, the majority of which did not show plastic responses. Notably, in no case was the interaction between selection regime and ethanol exposure significant after controlling for multiple comparisons, indicating that adaptation to ethanol in the E and M populations did not involve substantial changes in gene expression plasticity. The results give evidence that expression plasticity evolves considerably more slowly than mean expression.