Issues of cost and complexity have limited the study of the population sizes of men who have sex with men (MSM) and injection drug users (IDUs), two groups at clearly increased risk for human immunodeficiency virus (HIV) and other acute and chronic diseases. We developed a prototypical, easily applied estimation model for these populations and applied it to Miami, Florida. This model combined HIV prevalence estimates, HIV seroprevalence rates, and census data to make plausible estimates of the number and proportion of MSM and IDUs under a number of assumptions. Sensitivity analyses were conducted to test the robustness of the model. The model suggests that approximately 9.5% (plausible range 7.7%-11.3%) of Miami males aged 18 years or older are MSM (point estimate, N = 76,500), and 1.4% (plausible range 0.9%-1.9%) of the total population aged 18 years or older are IDUs (point estimate, N = 23,700). Males may be about 2.5 times more likely than females to be IDUs. The estimates were reasonably robust to biases. The model was used to develop MSM and IDU population estimates in selected urban areas across Florida and should be replicable in other medium-to-large urban areas. Such estimates could be useful for behavioral surveillance and resource allocation, including enhanced targeting of community-based interventions for primary and secondary HIV prevention.