The BOB.1/OBF.1 protein is a B-cell-specific coactivator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of the Oct proteins. However, animals deficient for BOB.1/OBF.1 showed virtually normal expression of genes that contain octamer motifs in their regulatory regions. To identify new genes that are regulated by BOB.1/OBF.1, we took advantage of a previously described cell system. RNAs differentially expressed in a BOB.1/OBF.1-deficient pre-B cell line and a derivative of this cell line expressing a hormone dependent BOB.1/OBF.1-estrogene receptor (BobER) fusion protein were isolated. Using the cDNA representational difference analysis method we could identify myosin light chain 1 atrial (MLC1A) isoform as a gene regulated by BOB.1/OBF.1. MLC1A was so far unknown to be expressed in tissues other than muscle. Here we demonstrate that MLC1A is indeed expressed in mouse pre-B cells. Analysis of the expressed mRNA revealed an alternative 5′ promoter element and an alternative splice product, which had not yet been described for the murine gene. Cotransfection experiments with reporter constructs driven by the MLC1A promoter suggest that the regulation by BOB.1/OBF.1 is indirect. Consistent with this conclusion is the observation that transcriptional induction of the endogenous MLC1A gene by BOB.1/OBF.1 requires de novo protein synthesis.