Inorganic polyphosphate (PolyP) is a polymer of orthophosphate residues that is abundant to all organisms, including the brain (1,2). Recently, it has been shown that PolyP is released by astrocytes in a calcium-dependent manner and acts as a gliotransmitter via P2Y1 purinergic receptors (3). However, the localisation and mechanisms of release of PolyP from astrocytes are unclear. Using DAPI and a novel PolyP fluorescent indicators, we studied the location of PolyP in primary astrocytes and the mechanisms of release. PolyP was found to compartmentalise in vesicle-like structures in primary cultures of astrocytes. Using Lysotracker Red to visualise lysosomes and doing colocalisation analysis, we identified some of these vesicles as lysosomes (Mander’s coefficients: Mx=0.49±0.066, n=7; My=0.61±0.046, n=7). However, PolyP also compartmentalises in non-lysosomal PolyP-vesicles, possibly in ATP-containing vesicles as demonstrated through intensity profiles. Release of polyphosphate out of lysosomal vesicles could be stimulated by the calcium ionophore ferutinin (5 μM) (4) or the protonophore FCCP (1 μM, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (5). FCCP furthermore induced an increase in [PolyP] in some non-lysosomal PolyP vesicles, indicating a PolyP uptake in response to the increase of cytosolic PolyP due to lysosomal PolyP release. We also observed a redistribution of the polyphosphate fluorescence signal in astrocytes between different genetic cell models of PD (PINK1 KO, LRRK2 KO). Using student t-test to statistically compare the average vesicle area size revealed that lysosomes were significantly increased in LRRK2 KO astrocytes (103.6%, p < 0.00005, n=220) as were the non-lysosomal PolyP vesicles (75.9%, p < 0.0005, n=81) compared to WT, indicating that the vesicles in LRRK2 KO were swollen. Moreover, the [PolyP] in PINK1 KO non-lysosomal vesicles decreased in response to FCCP, whereas [PolyP] was observed to increase in some vesicles for both WT and LRRK1 KO.This study has shown that PolyP is partially located in lysosomes and partially in unidentified non-lysosomal PolyP vesicles. Lysosomes released PolyP into the cytosol in a calcium-dependent manner, while some non-lysosomal PolyP vesicles increased in [PolyP], likely responding to the increase in [PolyP]c by taking up PolyP. The increase in [PolyP] in non-lysosomal PolyP vesicles was not observed in PINK1 KO. Furthermore, lysosomes and PolyP vesicles were shown to be swollen in LRRK2 KO compared to WT, suggesting a novel characteristic for the LRRK2 KO PD model. Considering the significant redistribution of PolyP and vesicles in astrocytes from PINK1 KO and LRRK2 KO compared to WT, astrocytic PolyP-dependent glial transmission might be altered in these PD models.