TRPC6 cation channels have been implicated in pressure-induced depolarization leading to activation of L-type Ca²⁺ channels and myogenic constriction. Both DAG and 20-HETE may mediate TRPC6 activation. We therefore questioned whether both PLC/DAG/PKC and PLA₂/CYP4A/20-HETE pathways are crucial for development of myogenic tone. Mesenteric arteries from C57BL/6 wild type or conditional, smooth muscle-targeted L-type channel knock-out (CaV1.2^-/-) mice were pressurized and diameter measured. In Kreb's solution vessel diameter was 192±3 mm at 40 mmHg, 199±5 mm at 80 mmHg and 188±4 at 120 mmHg (n=46). In Ca²⁺-free Krebs' solution (+EGTA) the passive diameter was 193±3 at 40 mmHg, 235±4 at 80 mmHg (P<0.05 vs. ctrl.) and 243±4 at 120 mmHg (P<0.05 vs. ctrl.). Thus, myogenic tone (%) was 0.3±0.5 at 40 mmHg, 15.7±0.9 at 80 mmHg and 22.7±0.6 at 120 mmHg in C57BL/6 mice (n=46). The PLA₂ inhibitor AAOCF3 (5 mM; n=5), the CYP4A inhibitor HET0016 (10 mM; n=3) or the putative TRPC channel inhibitor SKF96365 (5 mM; n=5) did not inhibit myogenic tone. The PLC inhibitor U73122 (0.5 mM; n=5) reduced myogenic tone from 19.1±2.1 to 12.0±3.2 % at 80 mmHg and from 24.3±1.6 to 15.5±3.1 % at 120 mmHg (P>0.05). The PKC inhibitor BIM-X (1 mM; n=5) reduced myogenic tone from 19.4±2.6 to 16.7±3.6 % at 80 mmHg and from 25.8±2.8 to 20.7±3.1 % at 120 mmHg (P<0.05). The DAG lipase inhibitor RHC80267 (20 mM; n=9) increased myogenic tone, in a PKC- dependent manner, from 10.3±1.9 to 13.6±3.1 % at 80 mmHg and from 19.2±1.5 to 20.4±2.2 % at 120 mmHg (P>0.05). Myogenic tone in vessels from CaV1.2^+/+ control mice was abolished by 10 mM nifedipine (n=3), and it was absent in CaV1.2^-/- mice (n=2). Our data indicated that activation of L-type channels was crucial for development of myogenic tone, whereas PLC/DAG/PKC, PLA₂/20-HETE and TRPC cation channels seemed to play little or no role. Future experiments will address the mechanism for pressure-induced depolarization in mouse mesenteric artery.