Mullerian Inhibiting Substance (MIS) is a glycoprotein hormone required for male sexual organogenesis, which signals through a heteromeric complex of type I and type II receptors. MIS has also been shown to inhibit fetal lung development in vivo and in vitro, although the mechanism of this inhibition is unknown. Branching morphogenesis was studied in fetal rat lungs incubated with proteolytically activated MIS. MIS exposure resulted in reduced total lung bud number as well as lung perimeter length. To assess whether the MIS type II (MR II) receptor is expressed in fetal rat lung, total RNA was prepared from gestation day 18 to day 20 lungs and analyzed by slot blot hybridization with a radiolabeled MR II. The results revealed positive slot hybridization in fetal lung RNA. To confirm these results, RT-PCR was performed on multiple fetal rat lung RNA samples, using a set of oligonucleotide primers designed from the 3′ untranslated region of rat MR II cDNA. Results consistently revealed an RT-PCR product of the expected size that when cloned and sequenced was identical to rat MR II. These studies suggest that MIS is a negative modulator of fetal lung branching in vitro and that the mechanism of this MIS-induced inhibition might be mediated via the putative MIS type II receptor.