BACKGROUND: Ca(2+) oscillations are irregular and heterogeneous. RESULTS: The correlations between NFκB/STAT3-GFP transcription and Ca(2+) spike amplitude/cumulative spike duration are revealed by simultaneous monitoring in single cells and validated in cell population. CONCLUSION: Ca(2+) oscillations regulate transcription through Ca(2+) spike amplitude and cumulative spike duration. SIGNIFICANCE: How irregular Ca(2+) oscillations control transcription is crucial for understanding biological Ca(2+) signal-regulated events. Agonist-stimulated Ca(2+) oscillations are universally irregular in their kinetics. How irregular Ca(2+) oscillations dynamically regulate agonist-stimulated downstream events has not been studied. To overcome the obstacles of irregularity and heterogeneity of Ca(2+) oscillations, agonist-stimulated Ca(2+) signaling and NFκB/STAT3-GFP nuclear translocation were simultaneously monitored in each single cell examined. The cause-effect relationship between Ca(2+) oscillation parameters and transcriptional activities was validated in cell populations through irregular Ca(2+) oscillations with varied parameters. The time duration of cumulative Ca(2+) elevations reaching the threshold Ca(2+) level for a transcriptional factor activation and Ca(2+) spike amplitude was found to control agonist-stimulated transcription and gene expression.