Cyclic GMP-dependent protein kinases (PKG) constitute a small family of enzymes that are encoded by two genes. Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated as Iα and Iβ. There are increasing evidences that PKG can play an important role in the inhibition of cell proliferation and induction of apoptosis. In our previous studies, the inhibitory effects of cGMP/PKG on the cell growth were indicated using breast cancer cell lines. Accordingly, the present study was designed to compare the expression levels of three PKG isoforms in normal, benign, and malignant breast tissues. The expression level of PKG isoforms was assayed using quantitative real-time RT-PCR. The correlation between relative expression of PKG isoforms and clinicopathological characteristics were also analyzed. Downregulation of PKG isoforms was observed in the malignant and benign tumors when compared to those of respective normal tissues. No significant correlation was found between PKGIα, PKGIβ, and PKGII expression and clinicopathological features. The present study is the first to evaluate the expression level of PKG isoforms PKGIα, PKGIβ, and PKGII in the malignant and benign breast tumors. Reduction in the PKG expression is an important evidence to support the antitumor activity of this enzyme in vivo.