Background: Feeding intolerance is a common issue in the care of preterm neonates. The condition manifests as delayed gastric content emptying and represents a therapeutic challenge, since the factors accounting for its manifestations are unknown. The main goal of this study was to comparatively investigate the age-related rat gastric and pyloric smooth muscle function and their putative regulators. We hypothesized that a reduced gastric muscle contraction potential early in life, contributes to the delayed gastric emptying of the newborn. Methodology: Newborn and adult rat gastric (fundal) and pyloric sphincter tissues were comparatively studied in vitro. The tissue-specific dissociated smooth muscle cells (SMC) shortening properties were evaluated and the key regulatory proteins Rho-associated kinase 2 (ROCK-2) and myosin light chain kinase (MLCK) expression determined. Results: Adult gastric and pyloric SMC shortening was significantly greater, when compared with the respective newborn counterpart. MLCK and ROCK-2 expression were developmentally regulated and increased with age. The newborn pyloric sphincter muscle expresses a higher neuronal nitric oxide synthase and phosphorylated vasodilator-stimulated phosphoprotein content, when compared with adult tissue. Conclusions: As compared with later in life, the newborn rat gastropyloric muscle has a Ca2+-related reduced potential for contraction and the pyloric sphincter relaxation-dependent modulators are overexpressed. To the extent that these rodent data can be extrapolated to humans, the neonatal delayed gastric emptying reflects the reduced stomach muscles contraction potential, as opposed to increased pyloric sphincter tone.