Fetal lung explant culture has been used extensively to study the regulation of expression of the human surfactant protein (SP) genes. In the present studies, explants from twenty, second trimester fetal lungs were used to study regulation of the human SPA genes, SPA1 and SPA2, by 100 nM dexamethasone (Dex), 30 ng/ml interferon γ (IFN γ), 0.1 mM cyclic 3′5′ adenosine monophosphate (cAMP), and 10 ng/ml tumor necrosis factor α (TNF α). Northern blots of total RNA isolated from explants that were cultured in the presence or absence of these compounds were hybridized with 1) an oligonucleotide common to both genes to study the effect on total SPA mRNA, 2) genespecific oligonucleotide to study the response of the individual genes to the compounds, and 3) an actinspecific oligonucleotide, used for normalization of RNA load. The SPA genotypes of the twenty fetal lungs were also determined using gene and allelespecific hybridization. The level of total SPA mRNA in control culture (Waymouth's medium) varied widely among the explants (coefficient of variation = 0.82) and was not correlated with the gestational age of the fetal lungs. The level of total SPA mRNA was, in general, increased by interferon γ (10/13) and cAMP (12/16), and decreased by TNF α (8/11) and Dex (11/13). The effect of the various treatments on the ratio of SPA1 to SPA2 mRNA was more complex. In all cases, Dex 100 decreased the ratio, indicating that SPA1 alleles are more inhibited by Dex than SPA2 alleles. For IFNγ, TNFα, and cAMP, the ratio varied among tissue samples, suggesting that the response of the different SPA1 and SPA2 alleles to these treatments varies. Supported by NIH HL49823.