Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae.