Factors positively influencing surfactant homeostasis in general and SP-B expression in particular are considered of clinical importance regarding an improvement of lung function in preterm infants. The objective of the study was to identify effects of physiological levels of caffeine on glucocorticoid-mediated SP-B expression in vitro and in vivo. Levels of SP-B and pepsinogen C were quantified by qPCR or immunoblotting in NCI-H441 cells daily exposed to caffeine and/or DEX. In vivo, SP-B expression was analyzed in BAL of preterm sheep exposed to antenatal DEX and/or postnatal caffeine. If DEX and caffeine were continuously present, SP-B mRNA and protein levels were increased for up to 6 days after induction (p<0.05). Additionally, caffeine enhanced SP-B mRNA expression in DEX-pretreated cells (p<0.05). Moreover, caffeine amplified DEX-induced pepsinogen C mRNA expression (p<0.05). After short-term treatment with caffeine in vivo, only slightly higher SP-B levels could be detected in BAL of preterm sheep following antenatal DEX, combined with an increase of arterial oxygen partial pressure (p<0.01). Our data demonstrated that the continuous presence of caffeine in vitro is able to amplify DEX-mediated SP-B expression. In contrast, short-term improvement of lung function in vivo is likely to be independent of altered SP-B transcription and translation. An impact of caffeine on release of surfactant reservoirs from lamellar bodies could however quickly affect SP-B content in BAL, which has to be further investigated. Our findings indicate that caffeine is able to amplify main effects of glucocorticoids which result from changes in surfactant production, maturation, and release.