The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.