Mitogen-stimulated human peripheral blood mononuclear cells (H-PBMCs) are conventionally used to culture primary human immunodeficiency virus type-1 (HIV-1) isolates in vitro. In this study, we attempt to increase the quality of primary HIV-1 stocks harvested from H-PBMC culture using medium replenishment procedures. Experimental/analysis results indicate that more frequent medium replenishment may not lead to improved quantity and quality of harvested virus stock titers,as determined by the viral core (p24) antigen content, viral infectivity, and viral particle-to-infectious unit ratio. This finding implies the conditioning factor(s) present in H-PBMC culture may be important for primary HIV-1 production. The optimal rate for intermittent medium replenishment to achieve the lowest viral particle-to-infectious unit ratio is around 0.25 volume/volume/day.