Two pathways control glucose-induced insulin secretion. A triggering pathway involves closure of K_ATP channels in the b-cell membrane, depolarization, influx of Ca²⁺ and rise in cytosolic [Ca²⁺]c, which then triggers exocytosis of insulin granules. An amplifying pathway augments insulin secretion without increasing [Ca²⁺]c further. It is not known how this amplification augments the efficacy of Ca²⁺ on exocytosis. In this study we tested a hypothesis, put forward by others, suggesting that amplification results from the acceleration of insulin granules translocation from a reserve pool to the exocytotic sites in the plasma membrane. Such translocation is achieved by granule movement along microtubules followed by their transfer across a sub-membrane web of microfilaments. Mouse islets were thus treated with agents known to interfere with microfilament function (Cytochalasin B, Latrunculin B or Jasplakinolid), with microtubule function (Nocodazole or Taxol) or both (Taxol and Cytochalasin B). The amplifying effect of glucose was tested by comparing insulin secretion and [Ca²⁺]c in low (1–3mM) or high (15mM) glucose after [Ca²⁺]c was raised by a high concentration of Tolbutamide (K_ATP channels closed) or by KCl in the presence of Diazoxide (K_ATP channels open). Depolymerization or stabilization of microfilaments increased Tolbutamide- or KCl-induced insulin secretion in low glucose and did not alter the amplifying action of high glucose. Depolymerization or polymerization of microtubules slightly inhibited insulin secretion in low glucose and unexpectedly caused a small inhibition of the [Ca²⁺]c rise. However, this did not impair the amplifying effect of high glucose (except for a slight attenuation by Taxol in the presence of KCl). Amplification of insulin secretion was still observed when both microfilaments and microtubules were disrupted by the combination of Cytochalasin B and Taxol. Our results show that metabolic amplification does not require an intact cytoskeleton, and do not support the hypothesis suggesting that the process corresponds to insulin granules mobilization from a reserve pool.