An increase in the intracellular calcium ion concentration ([Ca2+]i) controls a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Calcium signalling patterns can occur as single transients, repetitive oscillations or sustained plateaux, but it is not known whether these patterns are responsible for encoding the specificity of cellular responses. We report here that the amplitude and duration of calcium signals in B lymphocytes controls differential activation of the pro-inflammatory transcriptional regulators NF-kappaB, c-Jun N-terminal kinase (JNK) and NFAT. NF-kappaB and JNK are selectively activated by a large transient [Ca2+]i rise, whereas NFAT is activated by a low, sustained Ca2+ plateau. Differential activation results from differences in the Ca2+ sensitivities and kinetic behaviour of the three pathways. Our results show how downstream effectors can decode information contained in the amplitude and duration of Ca2+ signals, revealing a mechanism by which a multifunctional second messenger such as Ca2+ can achieve specificity in signalling to the nucleus.