Lymphocytotoxic alloantisera were obtained by cross immunization among outbred guinea pigs from a closed colony. Groups of antisera recognizing the same specificities were established by panel analysis, cross immunization among appropriate phenotypes, and absorption studies. Three antigens designated B1, B2, and B3 (perviously B, C, and D) were detected in our outbred colony and also found to be present in a wide variety of guinea pig strains. These antigens were shown by population and breeding studies to be allelic gene products of a first guinea pig locus (GPLA) (designated locus B). Four new guinea pig families homozygous for their B locus antigens are now in the seventh generation of inbreeding. NIH guinea pig strains 2 and 13, which share the B1 antigen, differ by a number of other antigens which have been designated I1, I2, I3 (previously A), and I4. These antigens differ from the B locus gene products in their molecular size and tissue distribution and it is, therefore, postulated that they represent an equivalent of the Ia antigens in mice (designated I region). Cross immunizations among inbred or outbred guinea pigs identical for currently known B locus and I region antigens yield further antisera, possibly recognizing additional Ia specificities and/or a second GPLA locus designated locus S. Whereas the conventional eosin lymphocytoxicity technique easily permits the detection of the widely distributed antigens (such as the B locus antigens), a more sensitive 51Cr release technique is required to demonstrate the "Ia equivalent" antigens of the guinea pig, possibly due to their restricted presence on the membrane of only some lymphoid cell populations.