The effect of heat treatment of milk on the formation of acid gel was examined using confocal scanning laser microscopy and low-amplitude dynamic oscillation throughout acidification. Milk samples were reconstituted by mixing colloidal phase from unheated or preheated skim milk, labeled with rhodamine B isothiocyanate, with the aqueous phase from unheated or preheated milk, labeled with fluorescein isothiocyanate. Gels were made by acidification with glucono-delta-lactone. The presence of material from preheated milk, that is, either the colloidal or the aqueous phase or both, led to an increase in the gelation pH and in the final elastic modulus and to a more branched network with larger pores. During acidification, the heat-induced serum complexes and the casein micelles did not appear to form separated gels with time or in space. Moreover, the colocalization in the final network of serum heat-induced complexes and casein micelles is particularly well observed in the presence of an aqueous phase obtained from preheated milk. Finally, because the rheological and microstructural properties of acid gels containing either micelle-bound or serum heat-induced complexes were similar, it was suggested that the serum heat-induced complexes interacted with the casein micelles early in the course of acidification and that formation of the network did not differ significantly whether the heat-induced complexes were initially found in the aqueous phase of milk or bound to casein micelles.