In heart, pore-forming Kv4 alpha channel subunits underlie the transient outward potassium current (Ito). Calcium signaling and activation of the nuclear factor of activated T-cells (NFAT) transcription factor are known to underlie regional variations of Ito expression (1-2), where nuclear localization of NFATc3 suppresses Kv4 expression. Exposure to elevated glucose activates NFATc3 in vascular smooth muscle (3). Here we tested the hypothesis that elevated glucose activates NFAT and attenuates Ito in murine cardiac muscle. Elevated levels of circulating glucose were measured in a murine model of diabetes (db/db) (db/db 47.79 ± 2.08 mM, n = 38 vs db-control 9.61 ± 1.34 mM, n = 4, p < 0.05). Cardiac Ito recorded from db/db myocytes was smaller than from db-control myocytes (at + 40 mV, 18.10 ± 1.91 pA/pF, n = 8 vs 31.21 ± 6.28 pA/pF, n = 8, p < 0.05). Exposing freshly dissociated murine cardiac myocytes (24 hrs) to elevated levels of D-glucose (HG-D, 25 mM) compared to normal levels (NG, 5.5 mM) reduced Ito in wild type (WT) myocytes (at + 40 mV, NG 56.15 ± 11.31 pA/pF, n = 6 vs HG-D 27.90 ± 5.20 pA/pF, n = 7, p < 0.05). However exposure to HG-D had no effect on Ito in myocytes from animals lacking NFATc3 (at + 40 mV normalised, NG 100 ± 2.29 %, n = 6 vs HG-D 122.107 ± 3.59 %, n = 3, p > 0.05). Enhanced nuclear localisation of NFATc3 was observed in db/db myocytes and in WT myocytes exposed to HG-D, however, not in WT myocytes exposed to hyperglycaemic conditions using the non-metabolisable enantiomer of D-glucose, L-glucose. Exposure of db/db myocytes to normal glucose levels (5.5 mM) resulted in reduced L-type calcium channel current (ICa(L)) (at 0 mV, HG-D 11.13 ± 0.67 pA/pF, n = 5, vs NG 7.48 ± 1.04 pA/pF, n = 4, p < 0.05). Conversely exposure of WT myocytes to HG-D increased the magnitude of ICa(L) (at 0 mV, NG 5.45 ± 0.35 pA/pF, n = 19 vs HG-D 7.46 ± 0.56 pA/pF, n = 11, p < 0.05). Examination of Cav1.2(alpha1C) Ser 1928 phosphorylation levels revealed exposure to HG-D produced increased Ser 1928 phosphorylation. In summary, we demonstrate, for the first time, the involvement of the NFAT signalling pathway in reduced cardiac Ito seen in response to elevated levels of glucose. In fact, reduced Ito with exposure to elevated levels of glucose may prove to be a key factor underlying the increased incidence of cardiac arrhythmias observed in the pathology of diabetes. Data are expressed as mean ± S.E.M. and where shown p values result from t-tests.