Although abnormal sarcoplasmic reticulum (SR) Ca2+ handling may cause heart failure, there has been no method to directly measure Ca2+ concentration in SR ([Ca2+](SR)) of living cardiomyocytes. We have measured [Ca2+](SR) by expressing novel fluorescent Ca2+ indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1-and Mer-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca2+](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium. (C) 2004 Elsevier Inc. All rights reserved.