Experiences of odours at meals are thought to affect food preference in many animals. To study appetite modulation by odours, we established a new experimental system based on the modification of a previous method, where flies fed sucrose flavoured with D-limonene subsequently showed reduced appetite to plain sucrose. In this new experimental system, a fly population was divided into two groups: (1) the "simultaneous" group of flies was exposed to D-limonene and sucrose simultaneously for 10 min, and (2) the "separate" group of flies was exposed to sucrose alone for 10 min, and 1h later, to limonene for 10 min. The appetite of flies in the "separate" group for sucrose was unaffected by the experiment, but the appetite of flies in the "simultaneous" group was significantly decreased, and this effect lasted for > or = 3 days. To investigate if this appetite modulation by D-limonene was based on long-term memory formation (protein synthesis), we examined the effects of the protein synthesis inhibitor, cycloheximide. Injection of cycloheximide 1h after exposure to limonene and sucrose inhibited the appetite suppression in the "simultaneous" group of flies. In addition, to exclude the possibility that D-limonene exerted its effect through taste, rather than odour, we examined the effect of removing of the olfactory organs, antennae and maxillary palps on appetite modulation by D-limonene. When the olfactory organs were removed, no reduction in appetite was observed in the flies in the "simultaneous" group, indicating olfaction. In our new and effective appetite-modulation system for flies, modulation of appetite by olfactory detection of D-limonene was shown.