Neutralizing antibodies (NAbs) can inhibit gene transfer by adenovirus in preclinical models, but this has not been confirmed in human trials, which have mostly utilized local delivery. Heretofore, it has not been studied whether intraperitoneal administration of adenovirus to ovarian cancer patients results in induction of NAbs in ascites or serum. Also, it is not known how ascites and serum NAbs correlate, or if preexisting ascites NAbs block gene transfer in humans. Before treatment, 33% of patients had an NAb titer of > 1000. Good correlation existed between serum and ascites NAb titers before (p = 0.0003) and after (p = 0.0008) treatment, and serum and ascites NAb titers were rapidly induced after treatment. Transgene expression was affected but not prevented by preexisting NAbs. Total anti-adenoviral antibodies did not correlate well with NAbs and the administered dose did not affect the level of NAb induction. Although serum and ascites NAbs correlated well, serum total protein concentration was over 4-fold higher, suggesting selective accumulation of proteins in ascites. These results imply that serum NAbs can be used as a surrogate for ovarian cancer-associated ascites NAbs. Also, although NAbs did not prevent gene transfer, it could be useful to attempt removal of ascites before intraperitoneal adenoviral therapy.