Microtubules in the diabetic heart are resistant to the depolymerising agent colchicine (Howarth et al. 2002). Here we examine the functional relevance of these colchicine-stable microtubules, using a high concentration of nocodazole (NOC) which disrupts stable microtubule populations (Webster & Patrick, 2000). Type 1 diabetes was induced by injection of streptozotocin (STZ; 60 mg kg^-1 i.p.) in male Wistar rats. 8-12 weeks later, animals were killed humanely, and ventricular myocytes isolated by enzymatic digestion. Myocytes were incubated with vehicle (0.2% DMSO) or 33 μM NOC for 1-3 h. The impact of NOC on microtubule density was assessed in fixed myocytes labelled with an antibody to β-tubulin. The effect of NOC on shortening and the [Ca²⁺]_i transient (using fura-2) was studied at 36°C (1 Hz stimulation). Statistical analysis was performed using the Student’s t test or the χ² test. Microtubule density was 10% higher in myocytes from STZ rats (P<0.05) compared with controls. Myocytes from STZ rats showed a tendency for shortening and [Ca²⁺]_i transient amplitude to be reduced (P>0.05). Time to peak [Ca²⁺]_i was increased (P<0.05). NOC decreased microtubule density in both populations of myocytes, but the effect was greater (P<0.01; χ² test) in control cells (mean density reduced by 30.7%; n=38-47) than in cells from diabetic rats (mean density reduced by 13.4%, n=56-54). Although the degree of microtubule disruption by NOC was less in myocytes from diabetic rats than controls, the functional consequences of microtubule disruption were much greater. In the presence of NOC, shortening increased by 45.0% in myocytes from STZ rats compared with 4.4% in controls (P<0.001; n=18-22; χ² test). Likewise, the amplitude of the [Ca²⁺]_i transient increased by 24.3% in myocytes from STZ rats compared with 6.0% in controls (P<0.001, n=19-21; χ² test). The prolonged time to peak [Ca²⁺]_i was absent in myocytes from diabetic animals after treatment with NOC. Microtubule disruption by NOC has a marked positive inotropic effect in myocytes from diabetic animals which is not seen in controls. This suggests that changes in the cardiac microtubule cytoskeleton are functionally relevant in the STZ model of diabetes. However, a distinction can be drawn between microtubules in STZ-treated animals, which are reported to be hypotensive, and those with hypertension-induced hypertrophy (Tsutsui et al. 1994) where microtubule disruption modifies shortening in the absence of changes in [Ca²⁺]_i.