Abstract： In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%±5.9% versus 57.9%±3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%±3.8% versus 24.0%±2.3%, P<0.0001; third round, 2.3%±1.6% versus 8.6%±1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200±23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1±10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.