The amounts and concentrations of secretin in extracts of rat and guinea pig small intestine were measured with a porcine secretin radioimmunoassay. Immunoactivity in the extracts comigrated with porcine secretin in sodium dodecyl sulfate acrylamide gel electrophoresis. In the small intestines of adult rats and newborn and adult guinea pigs, there was a marked proximal-to-distal gradient in secretin concentration; newborn rats exhibited approximately equal proximal and distal secretin concentrations. Concentrations were as follows: most proximal newborn or adult guinea pig segments, 80 ng/mg prot; most distal newborn or adult guinea pig segments, 2 ng/mg prot; most proximal adult rat segments, 50 ng/mg prot; most distal adult rat segments, 8 ng/mg prot; and newborn rat intestine, 11 ng/mg prot. In either rats or guinea pigs, the secretin concentration in the proximal small intestine fell between days 2 and 6 postpartum to levels 3- and 10-fold below their respective newborn values. It is consistent with the relatively mature state of the newborn guinea pig digestive tract that it displays the proximal-to-distal gradient of secretin that is characteristic of both adult guinea pigs and rats. In the proximal intestine of both species, intestinal growth outpaces the accumulation of secretin.