Seed germination and seedling establishment are the most critical periods among the developmental morphology stages in most plant species. Plant species diversity could be seen from the balance between the soil seed bank composition and the safe site availability. In Pampean grasslands of Argentina, plant species diversity is threatened by the advance of agriculture. Most of the original grasslands are lost as a result. In this context, it is increasingly important to know the mechanisms involved in the maintenance of plant diversity. Seedling emergence would be partly responsible for determining plant species diversity, and it may be determined by microsites conditions (competition level, ground cover, disturbances). We analyzed (i) the effect of above- and below-ground competition and litter application on seedling emergence in ungrazed areas, and (ii) the effect of vegetation cover, litter or dung, bare ground, and soil removal on seedling emergence in grazed areas. Within an exclosure to domestic livestock and a grazed site, different microsites were created, and natural seedling emergence was followed during two years. Above- and below-ground competition reduced grass emergence in ungrazed conditions, but did not affect forb emergence. Litter had no effect on emergence,although it reduced grass survival. At the grazed sites, soil disturbance had a positive effect on emergence of seedling of all vegetation types, while litter application showed a positive effect on grass emergence. However, survival in this area was very low (<10%). Competition reduced seedling emergence, showing to be a major control factor for the regeneration of the dominant species in the ungrazed sites. Litter presence reduced survival of grasses, but not of forbs. At the grazed sites, presence of litter improved microsites conditions, increasing grass emergence; at the same time, soil removal increased emergence of seedlings of all vegetation types, probably by favoring root development. In natural grasslands, availability of safe sites has an important role in the establishment of new individuals and plant species regenerations.