One of the major restrictions on the production of adenoviral vectors (AdV) is the so-called "cell density effect", i.e., a drop in cell-specific AdV productivity concomitant with increased cell concentration at infection (CCI) above 1 x 10(6) cell/ml. Although this "cell density effect" has been considered by several authors as a consequence of nutrient depletion and/or accumulation of byproducts, this study shows that the cell fraction at the S phase of the cell cycle may also play a role on AdV production at high CCI in 293 batch cultures. By infecting cells obtained from different growth stages at cell densities of 0.7 (lag), 1.6 (middle exponential), 3.3 (late exponential) and 5.0 x 10(6) (stationary) cell/ml, consisting of a descending proportion of cells in the S phase of the cell cycle in equalized cell density condition, it is shown that cell-specific AdV productivity is dependent on the cell cycle phase at infection at both the optimal CCI (1 x 10(6) cell/ml) and high CCI (4 x 10(6) cell/ml). In particular, under the equalized cell density conditions, the infection of cells with an S phase proportion of 50% at the high CCI gave the higher AdV volumetric productivity. Moreover, the lowest cell-specific AdV productivity was obtained with the lowest S phase proportion of 28% for both CCIs. These results indicate that a decrease in the proportion of cells at S phase at the time of infection also contributes to a decreased cell-specific AdV productivity at high cell densities. (c) 2006 Elsevier Inc. All rights reserved.