OBJECTIVES: T lymphocytes (T cells) are crucial important types of immune cells which are responsible for adaptive immune response during the progression of bacterial infectious diseases. Different subtypes of T cells can participate in regulating the progress of human periapical lesions. This study investigates the co-localization of different types of T cells by multiplex immunofluorescence staining (mIHC) from different types of human periapical lesions (PLs) which might elucidate the potential clinical relevance of T cell infiltration with PLs. MATERIALS AND METHODS: 20 PLs tissues and 6 healthy samples from healthy periodontal ligament tissues were collected for subsequent experiments. Histological examination and immunohistochemical analysis were performed using hematoxylin-eosin (H&E) staining, immunohistochemical staining (IHC) and mIHC. Data analysis involved quantifying the number of positive cells of CD3, CD4, GATA3, Tbet and Foxp3 using QuPath software. In addition, the co-localization of CD4, GATA3, Tbet and Foxp3 was also assessed. RESULTS: Radicular cysts (RCs) (n = 10) and periapical granulomas (PGs) (n = 10) were identified by histological evaluation. The number of CD3 CONCLUSIONS: This study demonstrates the different expression levels of T cells in PLs and indicates the distinct inflammatory states between the two types of PLs.