Abstract Background. The DNA methyltransferases (DNMTs) catalyze the addition of a methyl group to the 5-cytosine residue of CpG dinucleotides. This family of enzymes includes DNMT1 that functions as a DNA maintenance methyltransferase, and DNMT3A and DNMT3B that methylate previously unmethylated regions of DNA and are required for genome-wide de novo DNA methylation. However, there have been challenges to characterize DNMT3A protein expression in human tissue samples and it remains unclear its potential inhibition by classic and novel DNMT inhibitors. Materials and Methods. MCF-7 breast cancer and NCI-H23 non-small cell lung cancer cells were treated with DNMT inhibitors of 4'-thio-2'-deoxycytidine (T-dCyd), 4'-thio-5-aza-2'-deoxycytidine (aza-T-dCyd) and decitabine at 1 μM T-dCyd for 96h, and changes in DNMT3A expression were assessed by western blot and immunocytochemistry (ICC). DNMT3A expression was examined by immunohistochemistry in formalin-fixed and paraffin-embedded human breast, colorectal and lung cancers as well as lymphomas. Results. Expression of DNMT3A was decreased by all three DNMT inhibitors in MCF-7 and NCI-H23 cells detected by western blot and ICC. The mean staining index in NCI-H23 cells after T-dCyd treatment was 18.4, compared to 48.8 of vehicle control, by quantitative imaging and analysis (P <0.0001 by 2-sided t test). DNMT3A was primarily localized to the nucleus, and constitutively expressed in tumor cells at intermediate to high levels in 3.3% (2/60) of breast cancer, 0% (0/47) of colorectal cancer, 12% (9/75) of lung cancers, and 23% (34/148) of lymphomas across histology types including T-cell and diffuse B small cleaved/non-cleaved lymphomas as well as Hodgkin's disease. Additionally, DNMT3A expression was low in benign breast diseases (n = 16), normal colon tissues (n = 7) and normal lymph nodes (n = 11). Conclusions. The data demonstrate that DNMT3A is targeted for decrease by DNMT inhibitors and may serve as a pharmacodynamic and perhaps efficacy biomarker in early clinical trials. The intermediate to high levels of DNMT3A expression are rare in colorectal and breast cancers, and in 23% or fewer patients with either lymphomas or lung cancers. Citation Format: Sherry X. Yang, Dat Nguyen, Larry Rubinstein, Alice P. Chen, James H. Doroshow. DNA methyltransferase 3A expression in cancer cells following treatment with DNMT inhibitors and in human solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B12.