Cathepsin D (ctsd) is a lysosomal acid protease found in neutrophils and monocytes. We investigated whether differentiating agents increase the expression of ctsd mRNA in HL-60 cells. Treatment with either retinoic acid or calcitriol enhances the steady-state levels of ctsd mRNA in a dose-dependent manner. The stimulation by retinoic acid requires new protein synthesis. Pretreatment with retinoic acid enhances the response of the ctsd gene to prostaglandin E2. To determine whether the effects of retinoic acid and calcitriol are associated with differentiation, we pretreated Hl-60 cells for 120 h with inducers of granulocytic differentiation (lithium chloride, DMSO, and retinoic acid) and monocytic differentiation (calcitriol, sodium butyrate, and phorbol ester). Lithium chloride and DMSO do not significantly affect ctsd mRNA expression, and none of the granulocytic inducers alters the subsequent response of the ctsd gene to calcitriol. All of the monocytic inducers stimulate ctsd mRNA, and both calcitriol and sodium butyrate significantly potentiate the subsequent response to retinoic acid. Transcription initiation of the ctsd gene occurs at one major and several minor sites and is unaffected by treatment with retinoic acid and calcitriol or pretreatment with other differentiating agents. Although differentiation appears to influence ctsd mRNA expression, calcitriol and retinoic acid stimulate ctsd gene expression via mechanisms that are independent of their role in differentiation.