Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca²⁺ ([Ca²⁺]_i) elevation in smooth muscle cells (SMCs) via activation of its cognate A₁ receptors (A₁Rs). Mechanisms that underlie A₁R-dependent [Ca²⁺]_i elevation in renal vascular SMCs are not fully resolved. Whether A₁R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of A₁Rs by 2-chloro-N⁶-cyclopentyladenosine (CCPA) does not stimulate store-operated Ca²⁺ entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca²⁺]_i elevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca²⁺]_i concentration was unchanged in afferent arterioles isolated from newborn (0-day-old) pigs when compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca²⁺]_i in afferent arterioles from 20-day-old pigs. A₁R protein expression levels in the kidneys and afferent arterioles were unaltered in 0-day- versus 20-day-old pigs. However, TRPC3 channel protein expression level was ~ 92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of A₁Rs elicits receptor-operated Ca²⁺ entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of A₁Rs.