A rotation effect due to the inclusion of soybean ( Glycine max L. Merr.) in the continuous, no-till maize ( Zea mays L.) scheme in the Argentine Pampas region is known to exist. The magnitude and nature of the soybean contribution to N availability for the succeeding crop remains uncertain, however. This study sought to quantify the long-term (14 years) effects of the inclusion of full-season, no-till soybean in the continuous, no-till maize production scheme, on soil N availability indices and microbial activity of a silt-loam Luvic Phaeozem (Typic Argiudoll). Soil samples were collected at different stages of the maize over one growing season, and analyzed for nitrate-N, ammonium-N, protease, urease and microbial activities. Aerobic incubations were also performed to assess mineral N production. Higher nitrate-N, hydrolyzable-N, protease and microbial activities were detected in samples collected at maize flowering and harvest from the soybean–maize rotation than those from the maize monoculture. These results confirm the existence of larger labile N pools and higher capacity for N mineralization in soils under maize rotated with soybean than under maize grown in monoculture.