Thin ventricular trabeculae from rat hearts were skinned with the non‐ionic detergent Triton X‐100. The isometric force development of these muscles was investigated over a range of Ca2+ concentrations (0.2‐200 microM) in the presence of various concentrations of creatine phosphate (CP), creatine and inorganic phosphate (Pi). The addition of Pi (1‐30 microM) at constant ionic strength reduced the maximum Ca2+‐regulated force (obtained at 200 microM‐Ca2+). At 20 mM‐Pi force was 31 +/‐ 5% (mean +/‐ S.E. of mean, n = 7) of that in Pi‐free solution. This inhibitory action of Pi was not due to an inhibition of creatine kinase activity or to a reduction in the amount of free energy available for work from ATP hydrolysis. Increases in Pi concentration over the range 0‐20 mM shifted the sigmoid relationship between force and [Ca2+] to higher Ca2+ concentrations. The [Ca2+] required for 50% activation rose from 8.3 +/‐ 1.5 microM (mean +/‐ S.E. of mean, n = 7) in Pi‐free solutions to 19.8 +/‐ 4.2 microM in 20 mM‐Pi. An increase in CP concentration in the range 10‐30 mM had a small inhibitory effect on maximum Ca2+‐regulated force but did not alter the force‐‐[Ca2+] relationship. Creatine (0‐30 mM) was without effect on the skinned muscles. The inhibitory effects of Pi suggest that a net hydrolysis of CP to Pi and creatine in the myoplasm of intact cardiac cells could reduce force development at a given myoplasmic [Ca2+], especially if the latter was below the level needed to fully activate the myofibrils. This suggestion is discussed in relation to the CP hydrolysis and decrease in force development that are observed in cardiac muscle during hypoxia or ischaemia.