By using quantitative immuno-electron microscopy of two-sided labeled resin sections of rat exocrine pancreatic cells, we have established the relative concentrations of the secretory proteins amylase and chymotrypsinogen in the compartments of the secretory pathway. Their total concentration over the entire pathway was approximately 11 and approximately 460 times, respectively. Both proteins exhibited their largest increase in concentration between the endoplasmic reticulum and cis-Golgi, where they were concentrated 3-4 and 50-70 times, respectively. Over the further pathway, increases in concentration were moderate, albeit two times higher for chymotrypsinogen than for amylase. From trans-Golgi to secretory granules, where the main secretory protein concentration is often thought to occur, relatively small concentration increases were observed. Additional observations on a third secretory protein, procarboxypeptidase A, showed a concentration profile very similar to chymotrypsinogen. The relatively high concentration of amylase in the early compartments of the secretory route is consistent with its exceptionally slow intracellular transport. Our data demonstrate that secretory proteins undergo their main concentration between the endoplasmic reticulum and cis-Golgi, where we have previously found concentration activity associated with vesicular tubular clusters (Martínez-Menárguez JA, Geuze HJ, Slot JW, Klumperman J. Cell 1999; 98: 81-90).