Body. We tested the hypothesis that the negative functional effects of C-type Natriuretic Peptide (CNP) would be diminished in renal hypertensive (One-Kidney-One-Clip, 1K1C) cardiac hypertrophic rabbits and that this attenuated effect is due either to decreased cGMP production or to an altered downstream signaling pathway. Using isolated control and 1K1C ventricular myocytes, cell shortening data (video edge detection) were collected: (1) at baseline and after the addition of CNP 10 −8,−7 M, followed by KT8523, a cGMP-dependent protein kinase inhibitor; or (2) at baseline, following KT pretreatment and following subsequent CNP 10 −8,−7 M. In addition, cGMP levels were determined by radioimmunoassay at baseline and following CNP 10 −7 M. We found that in control myocytes, CNP decreased the percent shortening (5.7 ± 0.4 versus 4.0 ± 0.4%), maximal rate of shortening (58.7 ± 5.1 versus 45.2 μm/s) and maximal rate of relaxation (57.1 ± 4.9 versus 44.1 ± 3.4 μm/s) in a dose-dependent manner ( P < 0.05). All of these effects were reversed with subsequent KT administration. CNP failed to produce these effects in 1K1C myocytes. When pretreated with KT, CNP had no negative effect in both control and 1K1C myocytes. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes (63.6 versus 49.2 pmol/10 5 myocytes, P = NS); however, CNP produced a significant ( P < 0.05) rise in cGMP level in control (63.6 ± 7.8 versus 83.5 ± 11.3) but not in 1K1C (49.2 ± 2.6 versus 52.7 ± 5.6) myocytes. We conclude that in hypertrophic cardiac myocytes, the decreased negative functional effect of CNP is due to a decreased production of cGMP.