We have previously described T lymphocyte dependent guinea pig basophil growth from bone marrow precursors in vitro. In the current studies, basophil-stimulating factor (BSF) present in mitogen-stimulated splenic conditioned medium (CM) has been functionally characterized, utilizing an assay for BSF on nonadherent bone marrow target cells. BSF was found to be heat stable, nondialyzable, and inactivated by proteases. Monosaccharides known to inhibit guinea pig lymphokines yielded a unique profile of inhibition of BSF activity and nonidentity of BSF with guinea pig migration inhibition factor, interleukins 1 or 2, and granulocyte-macrophage colony-stimulating activity, from which BSF could be separated after gel filtration. BSF-containing CM also had no detectable interleukin-3 activity as measured in a murine assay. An inverse relation was found between interleukin 2 and BSF production by peritoneal exudate T cells (PEL) stimulated with antigen. Fractionation of serum-containing and serum-free CM demonstrated a molecular size for BSF of 50,000–65,000 daltons. Guinea pig BSF is a distinct T cell dependent lymphokine with an active protein moiety which may interact with target bone marrow cells through a cell surface carbohydrate receptor.