Objective:To determine the rate of development ofin vitroHIV resistance to (‐)2′‐deoxy‐3′‐thiacytidine (3TC) and relate the effect of dose to emergence of resistance.Methods:HIV‐infected men and non‐pregnant women, aged ≥ 18 years, with a CD4 count ≤ 300 × 106/l cells were fòllowed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma‐associated HIV RNA.Results:Phenotypic resistance was detected in approximately one‐third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV‐1 variants appeared reduced at high 3TC doses. Amino‐acid changes at codon 184 in HIV‐1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184.Conclusions:In vitroresistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild‐type virus, with regard to codon 184, in both patient plasma and PBMC.AIDS 1995,9:351‐357