Voltage-operated calcium channels (VOCCs) play a significant role in the regulation of intracellular calcium concentrations in cardiovascular, neuronal and skeletal tissues. Therefore, physiologically relevant screening methods for calcium channel modulators are required. A 45Ca2+ uptake assay based on clonal rat pituitary cell line GH4C1, possessing L-type VOCCs, was miniaturised into a 96-well plate format. The assay was validated by known Ca2+ channel blockers, verapamil and nimodipine (IC50 values 3.4 and 0.007 microM, respectively) and by a set of natural compounds and their synthetic derivatives. The results were consistent with our previous data and demonstrated the reliability of the assay. The signal-to-background ratio was 3.9 +/- 0.4, signal-to-noise ratio 10.3 +/- 2.3, Z' factor 0.59 +/- 0.10, and day-to-day variability in positive control values 5%. Furthermore, experiments were also made on a Biomek FX workstation to evaluate the suitability of the assay for automation. With minor modifications the assay is applicable, e.g. for studying possible Ca2+ channel activators in detail. The established 96-well plate assay modification for screening of calcium channel modulators reduces considerably the time, labour and resources needed for cell culture and experiments, and has significant advantages in terms of automation suitability and overall cost-efficiency.