Polytene chromosome studies were carried out on various population samples of Anopheles superpictus from different localities in Southern Italy. More than 7,000 female specimens, mostly obtained from daytime collections of indoor resting mosquitoes, were successfully scored for nurse cell polytene chromosomes. Night biting samples were also examined in some localities. Only one chromosomal polymorphism, due to a paracentric inversion involving the central third of the 2L arm, was recorded in all samples. In all localities, the inverted 2La arrangement showed remarkably stable frequency although the populations examined were isolated from each other and at least some of them have presumably been subject to bottle neck in recent years because of insecticide treatments or ecological changes affecting the availability of breeding places. Departures from the Hardy-Weinberg expectations, indicating an excess of heterokaryotypes, were noted and critically analysed by comparing samples obtained simultaneously in the same locality from different cow sheds, from different sections of the same cow shed and from night and day catches in the same cow shed. The phenomenon was not found uniformly distributed among indoor resting samples: significant departures from the Hardy-Weinberg expectations were observed in some cow sheds but not in others situated nearby or even adjacent to them. These results did not support the hypothesis that the excess of heterokaryotypes is due to their greater longevity or to differential mortality in the preimaginal stages. It is suggested that different karyotypes may react differently to microclimatic specific conditions, since the Hardy-Weinberg disequilibrium was mostly observed in samples from resting sites that were more lit and subject to wider climatic variation.