Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca² ([Ca² ]i); however, very few studies exist on [Ca² ]i dynamics in these cells. Other tissues display Ca² oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca² imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca² ]i changes of living germ cells in situ within the SST preparation. Ca² imaging revealed that a subpopulation of male germ cells display spontaneous [Ca² ]i fluctuations resulting from Ca² entry possibly throughout Ca_V3 channels. These [Ca² ]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca² fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca² oscillations for at least 10 min. Synchronous Ca² oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca² ]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca² signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.