In cardiac excitation-contraction (EC) coupling, calcium enters the cytosol via L-type Ca2+-channels (ICa), reverse Na+/Ca2+-exchange (NCXrev), or is released from the sarcoplasmic reticulum (SR) by Ca2+-induced Ca2+-release (CICR). The magnitude of calcium influx via the different pathways varies with the state of the cell. For example, in case of heart failure, CICR decreases and influx via NCXrev increases. We have developed a new method to quantify the calcium influxes in EC coupling. We used a combination of perforated patch clamp with fluorescence microscopy and mathematical modeling to study rainbow trout (Oncorhynchus mykiss) cardiomyocytes. First, the action potential (AP) of each cell was recorded at a frequency of 1.1 Hz. Second, we used the recorded AP to stimulate the cell in voltage clamp. ICa or NCX were inhibited to determine their respective currents with or without inhibition of SR. Fluorescence changes induced by Ca2+-transients were recorded by loading the cells with Fluo-4 AM. To estimate the contribution of ICa, NCX, and CICR, we composed a mathematical model of Ca2+-dynamics, which also takes into account intracellular buffering. For the given buffering constants, using spline approximated fluxes through ICa, NCX and CICR, and fluorescence gain as parameters, the model estimates the intracellular Ca2+ and fluorescence transients. Spline approximation of ICa and NCX fluxes can be found directly from the current measurements. CICR fluxes and other model parameters were found by least square fitting of the calculated and recorded fluorescence. Recordings of 27 different cells were used for analysis. In the control case, 9 cells were used to measure ICa and 6 cells to measure NCX current. When SR was inhibited, 6 cells were used to measure both ICa and NCX currents. Experiments were performed at 23 oC. Our data and analysis suggest that during in trout cardiomyocytes at the given temperature and frequency, the Ca2+-influxes are: 15-20% via L-type Ca2+-channels, 15-20% via NCXrev and 60-70% via CICR. When the SR was inhibited, we saw ~10% increased influx via L-type Ca2+-channels and ~40% increase via NCXrev compared to respective influxes in control case. The method is applicable to different species – including genetically modifiable mice and zebrafish – to study the cardiac functional phenotype under a range of physiological conditions.