p50/dynamitin (DM) is a major subunit of the microtubule-associated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1-91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150(Glued) subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1-91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate self-association of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.