The de novo purine biosynthetic pathway relies on six enzymes to catalyze the conversion of phosphoribosylpyrophosphate to inosine 5'-monophosphate. Under purine-depleted conditions, these enzymes form a multienzyme complex known as the purinosome. Previous studies have revealed the spatial organization and importance of the purinosome within mammalian cancer cells. In this study, time-lapse fluorescence microscopy was used to investigate the cell cycle dependency on purinosome formation in two cell models. Results in HeLa cells under purine-depleted conditions demonstrated a significantly higher number of cells with purinosomes in the G1 phase, which was further confirmed by cell synchronization. HGPRT-deficient fibroblast cells also exhibited the greatest purinosome formation in the G1 phase; however, elevated levels of purinosomes were also observed in the S and G2/M phases. The observed variation in cell cycle-dependent purinosome formation between the two cell models tested can be attributed to differences in purine biosynthetic mechanisms. Our results demonstrate that purinosome formation is closely related to the cell cycle.