Whether or not mechanisms underlying circadian locomotor rhythms and learning are related anatomically through the mushroom bodies (MBs) was investigated by monitoring behavioral rhythmicity in flies with MB lesions induced by chemical ablation and by mutations in five different genes. All flies tested were later examined histologically to assess (1) MB neuroanatomy, and (2) the condition of the putative pacemaker cells--the ventral Lateral Neurons (LN(v)s) and their terminals that project to the vicinity of the MB calyces. All groups of flies had normal rhythms except for mushroom body miniature (mbm; only in a wild-type Berlin genetic background) and mushroom body defect (mud). MB ablation had no effect on the gender-specific differences in the rhythmic activity profile that are typical of wild-type flies. However, ablated males had a slightly longer period than untreated males and were more active under constant dark conditions. LN(v)s and their arborization patterns appeared normal in MB-ablated and in most mutant flies. Activity defects of mbm flies were attributed to genetic background rather than to the mutation alone. Misrouted LN(v) projections and approximately 14% arrhythmia of mud flies were uncorrelated and attributed to pleiotropy rather than to specific effects of MB lesions. Our results imply that MBs are not involved in circadian activity rhythms but that they do have an inhibitory effect on activity levels of male flies.