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1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium‐sensitive fluorescent probe, Indo‐1. The relationship of the Indo‐1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo‐1 was assumed to be as slow as 10 s‐1, the descending limb (‘relaxation phase’) of the Indo‐1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo‐1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase‐plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo‐1 AM‐loaded cells. The Indo‐1 fluorescence‐length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i‐length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by ‘tetanization’ of cells in the presence of ryanodine is identical to the common [Ca2+]i‐length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate‐limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i‐length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady‐state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium‐sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)