Inversions of genetic segments during the evolution of Drosophila are well documented in the X chromosome and most autosomes, but little attention has been paid to chromosome 4, the smallest autosome or "dot chromosome" present in many Drosophila species. From our previous mapping we have defined probes that mark proximal, intermediate, and distal locations of chromosome 4 in D. melanogaster. In situ hybridizations on salivary gland polytene chromosomes with these probes show that the whole right arm, including genes within cytological region 101EF-102F, is inverted relative to D. simulans. We also used these probes to determine the orientation of the arm of the dot chromosome in nine species of Drosophila, including eight from the melanogaster subfamily. To account for the observed whole arm inversions of chromosome 4 in five of the nine species examined, we propose that three inversion events have occurred during the evolution of these species. These whole arm inversions may explain some of the unusual features of this chromosome.