Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue.The myosins constitute a superfamily of 18 classes (1 conventional and 17 unconventional) (1), all of which share sequence similarity in the motor domain. It is thought that they also share the ability to translocate along actin filaments. Myosins play roles in numerous cellular activities, such as division, endocytosis, cell movement, and organelle trafficking. In addition to the conserved motor domain, all myosins contain one or more conserved IQ motifs (consensus sequence IQXXXRGXXXR, where X is any amino acid) that bind calmodulin (CaM), 4 a ubiquitous calcium sensor protein, or CaM-like light chains (2-4). The ATPase activity and motility of unconventional myosins can be regulated by calcium binding to CaM or a CaMlike protein (5).Members of the myosin VI class appear to be unique among the unconventional myosins in their direction of translocation along actin. They move toward the pointed, or minus, end of actin filaments (6) whereas all other myosins tested move toward the barbed, or plus, end. Recent structural studies of pig myosin VI (7) indicate that this reversed directionality is associated with one of the two unique inserts in myosin VI, termed insert 2. This sequence is located between the converter region of the motor domain and the IQ motif, and its converter-proximal sequences are proposed to redirect the lever arm in the opposite direction relative to other myosins.Insert 2 is also proposed to perform a second, structural, role in myosin VI. In the pig crystal structure, its more C-terminal sequences form a continuous helix with the IQ motif, thus potentially exte...