Abstract: BACKGROUND: Cytoplasmic dynein mediates spindle positioning in budding yeast by powering sliding of microtubules along the cell cortex. Although previous studies have demonstrated cortical and plus-end targeting of dynein heavy chain (Dyn1/HC), the regulation of its recruitment to these sites remains elusive. RESULTS: Here we show that separate domains of Dyn1/HC confer differential localization to the dynein complex. The N-terminal tail domain targets Dyn1/HC to cortical Num1 receptor sites, whereas the C-terminal motor domain targets Dyn1/HC to microtubule plus ends in a Bik1/CLIP-170- and Pac1/LIS1-dependent manner. Surprisingly, the isolated motor domain blocks plus-end targeting of Dyn1/HC, leading to a dominant-negative effect on dynein function. Overexpression of Pac1/LIS1, but not Bik1/CLIP-170, rescues the dominant negativity by restoring Dyn1/HC to plus ends. In contrast, the isolated tail domain has no inhibitory effect on Dyn1/HC targeting and function. However, cortical targeting of the tail construct is more robust than full-length Dyn1/HC and occurs independently of Bik1/CLIP-170 or Pac1/LIS1. CONCLUSIONS: Our results suggest that the cortical association domain is normally masked in the full-length dynein molecule. We propose that targeting of dynein to plus ends unmasks the tail, priming the motor for off-loading to cortical Num1 sites.