Promyelocytic leukemia nuclear bodies (PML NBs) are dynamic nuclear organelles suggested to be involved in tumor suppression, viral defense, DNA repair, and transcriptional regulation. However, their precise role in one or more of these processes is still largely unknown. In order to gain additional insights into their potential functions, I performed biochemical and dynamic analysis of PML NBs in this study. I have developed a fractionation protocol that successfully enriched structurally intact PML NBs from the immunoprecipitation eluate of mouse tissues. The protein constituents in the eluate were further identified by mass spectrometry. The presence of many nuclear matrix proteins that were co-immunoprecepitated with PML protein support the idea that PML NBs may have a direct association with the nuclear matrix. To analyze the dynamics of PML NBs upon entry into and exit from mitosis, I developed a U2OS cell line stably expressing two different marker proteins that localize to PML NBs, PML-ECFP and EYFP-Sp100. Three dimensional time lapse live cell imaging revealed that PML NBs exhibit a higher percentage of rapid directed movement when cells progressed from prophase to pro-metaphase as compared to their interphase dynamics. This window of increased dynamic movement occurred upon nuclear entry of Cyclin B1 but prior to nuclear membrane breakdown. The PML NBs moved within the interchromatin space and fused to form fewer mitotic PML NBs which contain lower levels of Sp100 and Daxx protein as compared to interphase PML NBs. Upon exit from mitosis, Sp100 and Daxx entered the daughter nuclei after a functional nuclear membrane was reformed. However, the formation of PML NBs was dependent upon the presence of PML protein. My data suggest that entry into prophase results in a loss of tethering between regions of chromatin and PML NBs thereby resulting in their increased dynamics. Upon exit from mitosis, PML NB formation is initiated by PML protein. Overall, by using biochemical and cell biological approaches, my study suggests that PML NBs have a close association with nuclear matrix proteins and chromatin which likely influences their nuclear organization and function.