The HIV infection status of a cohort of 600 prospectively followed children born to HIV infected mothers was determined using HIV peripheral blood culture tests at 0, 3, and 6 months of age, HIV serology at ≥15 months, and CDC AIDS criteria. We estimated transmission rates using five methods which differed in how HIV indeterminates are handled. These methods were applied at two points in time to illustrate effects of length of follow-up of the cohort on results. In January 1997, 30 months after the last birth, transmission rate estimates ranged from 15.5% (known positives/known positives × known negatives) to 18.1% (known positives × those with one positive culture × deaths/entire cohort minus those lacking negative cultures at age ≥5 months). Estimates ranged from 14.8% to 20.7% using the subcohort of 284 children followed ≥12 months as of May 1993. These results indicate that methods for assigning HIV infection status and for handling HIV indeterminates should be carefully defined when estimating transmission rates.