The role of protein carboxyl methylation in amylase secretion from incubated rat pancreatic lobules was assessed. All the components of the carboxyl methylation system, protein carboxyl methylase, methyl-acceptor protein substrates, and protein methylesterase, were found in the rat pancreas. Protein carboxyl methylation is stimulated by both pancreozymin and carbachol at concentrations that are equally effective in stimulating amylase release. The secretagogue-enhanced increase in protein carboxyl methylation was concomitant with the onset of the secretory response. These responses were blocked by the receptor antagonists, dibutyryl cGMP and atropine. Inhibitors of protein carboxyl methylase inhibited carboxyl methylation stimulated by pancreozymin but did not affect the secretory response to the hormone. Ethyleneglycol-bis-(beta-aminoethylether)-N-N'-tetraacetic acid blocked protein carboxyl methylation and amylase release, indicating that extracellular calcium was essential for both processes. The presence or absence of calcium did not alter the enzyme activity in lobule homogenates. The data suggest that only a small part of the total cellular protein carboxyl methylation activity is directly involved in pancreatic enzyme secretion. Like the calcium requirement in secretion, protein carboxyl methylation also lies distal to the stimulant-receptor interaction.