Density-dependent pollen germination and tube growth in vitro is a well-documented phenomenon, termed the pollen population effect, but far less is known about its molecular basis. We present evidence to support phytosulfokine-alpha [Y(SO3H)IY(SO3H)TQ; PSK-alpha] as a native bioactive factor contributing to this effect. Mature pollen grains of Nicotiana tabacum L. var. macrophylla were incubated in liquid medium for 2 h. Pollen germination frequency increased in a density-dependent manner from 625 to 46,000 grains/ml. Conditioned medium, obtained from the medium of pollen cultured at a density of 10,000 pollen grains/ml for 12 h, promoted the germination of pollen cultured at a low density (625 grains/ml). A rabbit antiserum against PSK-alpha specifically inhibited the promotive effect of conditioned medium. Quantification by enzyme-linked immunosorbent assay showed that the conditioned medium contained 0.4 nM of PSK-alpha. Exogenous PSK-alpha also stimulated pollen germination in the low-density culture. These results indicate that PSK-alpha is an important regulator involved in the pollen population effect.