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Distribution of ink jet ink in paper substrates and the consequences of ink penetration for printing color reproduction have been studied by combining microscopic image processing with spectroscopic analysis. The study focused on the effects of the composition of uncoated paper, for five laboratory papers plus two commercial products, all consisting of similar pulps but with different combinations of additives. In particular, it was observed that hydrophobizing internal size agents significantly reduced ink penetration, while their effect on paper optical properties was negligible. This observation thus made it possible to study experimentally the pure effects of ink penetration. Pairwise comparisons of prints on such laboratory papers with identical optical properties revealed remarkable impacts of ink penetration on optical density, causing color saturation reduction and color shift. These experimental observations confirmed the theoretical predictions.