The expression levels of estrogen receptor 1 ( ESR1), arylamine N-acetyltransferase 1 ( NAT1), and arylamine N-acetyltransferase 2 ( NAT2) are implicated in breast cancer; however, their co-expression profiles in normal breast tissue, primary breast tumors and established breast cancer cell lines are undefined. NAT1 expression is widely reported to be associated with ESR1 expression and is frequently investigated in breast cancer etiology. Furthermore, the NAT2 phenotype has been reported to modify breast cancer risk in molecular epidemiological association studies. Understanding the relationships between the expression levels of these genes is essential to understand their role in breast cancer etiology and treatment. In the present study, NAT1, NAT2 and ESR1 expression data were accessed from repositories of RNA-Seq data covering 57 breast cancer cell lines, 1,043 primary breast tumors and 99 normal breast tissues. The relationships between gene expression, and between NAT1 activity and RNA expression in breast cancer cell lines were evaluated using non-parametric statistical analyses. Differences in gene expression in each dataset, as well as gene expression differences in normal breast tissue compared to primary breast tumors, and stratification by estrogen receptor status were determined. NAT1 and NAT2 mRNA expression were detected in normal and primary breast tumor tissues; NAT1 expression was much higher than NAT2. NAT1 and ESR1 expression were strongly associated, whereas NAT2 and ESR1 expression were not. Although NAT1 and NAT2 expression were associated, the magnitude was moderate. NAT1, NAT2, and ESR1 expression were increased in primary breast tumor tissue compared with normal breast tissue; however, the magnitude and significance of the differences were lower for NAT2. Analysis of NAT1, NAT2, and ESR1 expression in normal and primary breast tissues and breast cancer cell lines suggested that NAT1 and NAT2 expression are regulated by distinctive mechanisms, whereas NAT1 and ESR1 expression may have overlapping regulation. Defining these relationships is important for future investigations into breast cancer prevention.