Our laboratory has investigated replicating adenovirus-human sodium iodide symporter (Ad-hNIS) vectors in a combinatorial oncolytic approach known as radiovirotherapy. However, hNIS-mediated iodide sequestration requires an intact cell membrane, and the enhancement of infectivity may alter the radioiodide accumulation in vivo. To assess these effects, we constructed Ad-NIS vectors expressing NIS from the major late promoter. Viral tropism was altered using a hybrid Ad5/3 fiber, and rates of viral spread altered through expression of the Ad death protein (ADP). The hybrid 5/3 fiber enhanced Ad-mediated cytolysis and radioisotope uptake in vitro. Replicating ADP-lacking viral vectors showed levels of uptake similar to non-replicating vectors that declined as cells lysed. ADP expression enhanced the rate of cell lysis and viral release, but reduced the peak and duration of radioiodide uptake. SPECT-computed tomography imaging showed the Ad5/3-noADP-hNIS vector induced significantly more isotope uptake than other vector structures, indicating that viral spread may not always make up for the reduced NIS expression as in our work with prostate cancer. These results indicate that replicating, infectivity-enhanced Ad-NIS vectors provide superior overall efficacy, but also indicate that the effect of replication speed requires tumor and model-specific testing.