A cDNA encoding a new human actin-related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin-related protein was subsequently named ARP3β and represents the second example of multiple isoforms of an actin-related protein in a single organism. The ARP3β gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3β mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3β pseudogene was detected on chromosome 2p11 by database searching. Several ARP3β mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3β in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3β in the physiology of the development and/or maintenance of distinct subsets of nerve cells.