Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro-10 and ro-11. ro-10 and ro-11 are non-essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro-10 and ro-11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin-related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro-10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro-11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.