Using a fluorescent focus assay, complementation and interference effects of Ad2 and Ad41 on each other in mixed infection were investigated. Ad2 provided a helper function for Ad41 late antigen synthesis in cells normally non-permissive for Ad41 growth (HEF cells), and enhanced Ad41 late antigen synthesis in semi-permissive Chang conjunctival cells. The degree of complementation by Ad2 was dependent on its input concentration. In addition, interference by Ad41 on Ad2 replication was seen in HEF cells. The degree of interference by Ad41 was dependent on the relative time of infection by each serotype. The complementation results in HEF cells suggest an absolute dependency of Ad41 on an adenovirus helper function in these cells. The results presented are consistent with the postulated helper function provided in trans by 293 cells, which are transformed by Ad5 early region 1.