Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DC CD40LT), or monocyte-conditioned medium (DC MCM) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1 LAI or R5 HIV-1 Ba-L were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4 low (CXCR4 low) CCR5+ iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1 LAI entered more efficiently into DC CD40LT or DC MCM with up-regulated CXCR4. CCR5 low DC CD40LT still allowed entry of HIV-1 Ba-L, whereas CCR5− DC MCM displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1 Ba-L reverse transcription was more efficient than that of HIV-1 LAI, but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3. GFP or pNL4-3. Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60–75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-κB proteins and SP1 and SP3 transcription factors.