Cell populations independently recovered from low-temperature storage resulted in two general phenotypes with respect to HIV-1 3T5T long terminal repeat–green fluorescent protein (LTR-GFP) basal expression. TF-1, U-937, and Jurkat cells were stably transfected with the HIV-1 LAI LTR, which was placed in a GFP expression vector (pEGFP-N1) using the AMAXA Nucleofector System (Lonza, Basel, Switzerland). Mutagenesis was used to introduce the position 3 C-to-T change at CCAAT/enhancer binding protein (C/EBP) site I (3 T), a position 5 C-to-T change at Sp site III (5 T), or to introduce both the 3 T and 5 T variants into the same LAI LTR (3T5T). Flow cytometric analysis was used to measure the ability of each LTR to drive GFP expression as compared with their parental (WT) counterparts. The fluorescence patterns in untransfected cells are depicted in gray. The LAI WT LTR is shown in red; LAI 3 T LTR in green; LAI 5 T LTR in orange; and the HIV-1 LAI 3T5T LTR in blue. Phenotype expression pattern 1 is shown by panels on the left, while the phenotype expression pattern 2 is shown by panels one the right. All cells were analyzed by flow cytometry for basal levels of LTR-driven GFP expression between passages 6 and 8.