A size partitioning of several trace metals (Zn, Cd, Pb, Cu, Ni, Co, Mn, Fe and Al) between five size fractions (<3 kDa, 3 kDa–0.1 µm, 0.1 µm–1.2 µm, 1.2 µm–5 µm and >5 µm) was studied in the vertical salinity gradient of the highly stratified Krka River estuary. The results indicated a dominant river source for Zn, Co, Mn, Fe and Al and a diluting effect on Cd, Pb and Ni. The truly dissolved fraction (<3 kDa) dominated the Zn, Cd, Cu, Ni and Co pool, and a large part of Pb, Mn, Fe and Al was present in >5 µm particles. Pb, Mn, Fe and Al were closely related, showing a precipitation and colloidal aggregation in the surface layers and dissolution in the seawater layer. The highest percentage (30–37%) of colloids (3 kDa–0.1 µm) in the dissolved pool was found for Pb, Cu, Fe and Al. Differences in size distribution between low and high river flow periods revealed that Zn, Pb, Co, Mn, Fe and Al are introduced by the river mostly in the 3 kDa–5 µm size range. Therefore, a low percentage of colloidally bound metals compared to other coastal areas can be explained by a limited riverine input of terrigenous material, characteristic for this estuary. Correlation with PARAFAC components revealed associations of Cu with protein-like substances and Co with humic-like substances. The accumulation of Cu at the freshwater-seawater interface coupled with an increase of its colloidal fraction was observed, apparently governed by biologically produced organic ligands.