The effect of glucocorticoid on the regulation of pulmonary surfactant protein B (SP-B) synthesis was studied in a human pulmonary adenocarcinoma cell line. Northern blot analysis demonstrated a marked increase in SP-B mRNA expression after treatment with dexamethasone for 48 h. Actinomycin D, puromycin, or cycloheximide blocked the induction of SP-B mRNA by glucocorticoid. Nuclear run-on experiments demonstrated that the effects of dexamethasone on SP-B mRNA were due in part to increased transcription of the SP-B gene. However, during this time period, there was a discrepancy between SP-B gene transcription, which was increased only 2- to 4-fold, and SP-B mRNA, which increased 60- to 150-fold after treatment with dexamethasone. In the presence of actinomycin D, SP-B mRNA was relatively stable, decreasing slowly in the presence or absence of glucocorticoid. In contrast to the relative stability of SP-B mRNA in the presence of actinomycin D, SP-B mRNA was markedly decreased after exposure to puromycin, supporting the premise that continued protein synthesis, rather than transcription alone, is required for maintenance of SP-B mRNA levels. Induction of SP-B expression by glucocorticoids was dependent on enhanced SP-B gene transcription and was also dependent on continued protein synthesis. The discrepancy between the relative enhancement of SP-B transcription and SP-B mRNA suggests that posttranscriptional factors influence SP-B expression in this cell line.