Cytoplasmic dynein transports various cellular cargoes including early endosomes, but how dynein is linked to early endosomes is unclear. We find that the Aspergillus nidulans orthologue of the p25 subunit of dynactin is critical for dynein-mediated early endosome movement but not for dynein-mediated nuclear distribution. In the absence of NUDF/LIS1, p25 deletion abolished the localization of dynein-dynactin to the hyphal tip where early endosomes abnormally accumulate but did not prevent dynein-dynactin localization to microtubule plus ends. Within the dynactin complex, p25 locates at the pointed end of the Arp1 filament with Arp11 and p62, and our data suggest that Arp11 but not p62 is important for p25-dynactin association. Loss of either Arp1 or p25 significantly weakened the physical interaction between dynein and early endosomes, although loss of p25 did not apparently affect the integrity of the Arp1 filament. These results indicate that p25, in conjunction with the rest of the dynactin complex, is important for dynein-early endosome interaction.