In order to estimate the incidence of invasive pneumococcal diseases (IPD) and the amount of vaccine preventable serious infections, a 1-year population-based surveillance was undertaken in two comparable Italian regions (Piemonte and Puglia, representing 14% of the Italian population) prospectively collecting data and strains from all the hospital microbiological laboratories. A retrospective analysis of hospital discharge records, matched with the laboratory database, was also undertaken in nine hospitals in these two regions to determine the frequency of use of blood cultures and its impact on IPD incidence estimate. For children under 2 years of age, the incidence rates of IPD were 11.3 per 100,000 and 5.9 per 100,000 in Piemonte and in Puglia, respectively; for subjects 65 years of age and over the incidence rates were 5.7 per 100,000 and 0.2 per 100,000, in the two regions, respectively. The number of blood cultures performed was six times higher in Piemonte than that in Puglia. About 96% of isolates from IPD patients, aged 65 years and over, belonged to serogroups included in the 23-valent polysaccharide vaccine, whereas about 79% of strains isolated from patients under 5 years of age were related to serotypes included in the 7-valent conjugate vaccine. The estimate of the incidence of IPD is affected greatly by the different attitudes in performing blood cultures, especially in older patients. In Italy, bacteriological culture procedures should be undertaken more frequently to provide decision-makers with reliable estimates of serious vaccine preventable conditions.