Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 μm/s and over distances of less than 5 μm, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.