Information available on bloodstream infection (BSI) is usually restricted to short periods of time, certain clinical backgrounds, or specific pathogens, or is just outdated. We conducted the current prospective study of patients with BSI in a 1750-bed teaching hospital to evaluate workload trends and the incidence and etiology of BSI in a general hospital during the last 22 years, including the acquired immunodeficiency syndrome (AIDS) era. The main outcome measures were laboratory workload, trends in incidence per 1000 admissions and per 100,000 population of different microorganisms, and the impact of the human immunodeficiency virus (HIV) epidemic in the period 1985-2006.From 1985 to 2006 we had 27,419 episodes of significant BSI (22,626 patients). BSI incidence evolved from 16.0 episodes to 31.2/1000 admissions showing an annual increase of 0.83 episodes/1000 admissions (95% confidence interval, 0.61-1.05; p < 0.0001). The evolution of the incidence per 1000 admissions and per 100,000 population of different groups of microorganisms was as follows: Gram positives 8.2 to 15.7/1000 admissions and 66.8 to 138.3/100,000 population; Gram negatives 7.8 to 16.2/1000 admissions and 63.5 to 141.9/100,000 population; anaerobes 0.5 to 1.3/1000 admissions and 4.1 to 11.7/100,000 population; and fungi 0.2 to 1.5/1000 admissions and 1.7 to 12.5/100,000 population. All those differences were statistically significant. We observed the emergence of multiresistant Gram-positive and Gram-negative microorganisms. At least 2484 episodes of BSI (9.1%) occurred in 1822 patients infected with HIV. The incidence of BSI in HIV-infected patients increased from 1985 and reached a peak in 1995 (17.6% of BSI). Since 1995, the decrease was continuous, and in 2006 only 3.9% of all BSI episodes occurred in HIV-positive patients in our institution. We conclude that the BSI workload has increased in modern microbiology laboratories. Gram-positive pathogens have overtaken other etiologic agents of BSI. Our observation shows the remarkable escalation of some resistant pathogens, and the rise and relative fall of BSI in patients with HIV.