1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has been reported to stimulate lung maturity, alveolar type II cell differentiation and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)₂D₃ stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)₂D₃. 1,25(OH)₂D₃ significantly decreased SP-A mRNA in human fetal lung tissue, but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)₂D₃ alone had no significant effect on SP-A mRNA or protein levels, but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a non-ciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)₂D₃ had no significant effect on SP-B mRNA levels in lung tissue, but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)₂D₃ in lung tissue incubated ± cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)₂D₃ is not coordinated; 1,25(OH)₂D₃ decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.