The Spi-B transcription factor is an Ets protein expressed in B lymphoid cells and closely related to the Spi-1/PU.1 oncoprotein. By mutational analysis, we showed that Spi-B is phosphorylated by casein kinase II in vitro on four serine residues. Mutation of these four serines to alanines prevented the phosphorylation of Spi-B in vivo, increased the ability of Spi-B to transactivate expression of a reporter gene and led to a decrease of Spi-B stability. We propose that the phosphorylation of Spi-B may participate in the modulation of Spi-B functional activity by controlling its intracellular protein level.