In this study we considered the effect of chloroquine on the processing and intracellular distribution of internalized secretin radioligand in acinar cells. Chloroquine (100 μM) had no effect on the total amount of 125I-secretin bound but had marked effects on the processing of this radioligand in acinar cells. After an initial 60 min of radioligand binding in the presence and absence of chloroquine, cells were washed free of unbound radioligand, resuspended and then processed for different times at 37°C. During 60, 120 and 180 min of processing, the amount of internalized radioligand in the presence of 100 μM chloroquine was increased by 116, 194 and 273%, respectively, compared to untreated control samples. Chloroquine also increased the amount of intact 125I-secretin radioligand within the cell as measured by rebinding to pancreatic plasma membranes. After 120 and 180 min of processing, intact peptide within the acinar cell was 25 and 66% greater in the presence of this agent than in control samples ( P ≤ 0.01). To determine if chloroquine affected intracellular localization of the secretin radioligand, we measured the amount of radioactivity in soluble and particulate fractions of cell homogenates. Chloroquine decreased radioactivity entering particulate fractions of the cell by greater than 35% after 120 and 180 min of processing ( P ≤ 0.01). This study demonstrates that (1) chloroquine inhibits the intracellular degradation of secretin in acinar cells and (2) chloroquine alters intracellular localization of this peptide during processing.