Phospholipid (PL) scramblase is a 35 kDa protein that is thought to mediate Ca2+-induced bidirectional transbilayer movement of plasma membrane phospholipids in activated, injured, or apoptotic cells. We recently reported the molecular cloning of a PL scramblase of human (HuPLSCR1) and mouse origin, respectively. In the present study, the gene for HuPLSCR1 was cloned from a human genomic library. The gene size is 29.7 kb and includes nine exons. Analysis of the 5' flanking genomic sequence with luciferase reporter constructs located the promoter to a region spanning from -95 to +60 of the first (untranslated) exon. Furthermore, we report the molecular cloning of three additional novel cDNAs encoding proteins with high homology to HuPLSCR1. The predicted open reading frames encode proteins with 59% (HuPLSCR2; 224 aa), 47% (HuPLSCR3; 295 aa) and 46% (HuPLSCR4; 329 aa) identity, respectively, to HuPLSCR1. All members of the PLSCR gene family conserve those residues contained in the segment of the PLSCR1 polypeptide that was previously shown to bind Ca2+. With the exception of HuPLSCR2, these proteins also each contain multiple PXXP motifs and a PPXY motif located near the N-terminus, implying the potential for interaction with SH3 or WW domain-containing proteins, respectively. HuPLSCR1, 2, and 4 were found to be closely clustered on chromosome 3 (3q23), whereas HuPLSCR3 is located on chromosome 17. Northern blots revealed that the expression of HuPLSCR2 is restricted to testis, whereas HuPLSCR1, 3 and 4 are expressed in most of the 16 tissues examined. Notable exceptions were HuPLSCR4, which was not detected in peripheral blood lymphocytes, and HuPLSCR1 and HuPLSCR3, which were not detected in brain.