The directional distribution of the phytohormone auxin is essential for plant development. Directional auxin transport is mediated by the polarly distributed PIN-FORMED (PIN) auxin efflux carriers. We have previously shown that efficient PIN1-mediated auxin efflux requires activation through phosphorylation at the four serines S1-S4 in Arabidopsis thaliana. The Brefeldin A (BFA)-sensitive D6 PROTEIN KINASE (D6PK) and the BFA-insensitive PINOID (PID) phosphorylate and activate PIN1 through phosphorylation at all four phosphosites. PID, but not D6PK, can also induce PIN1 polarity shifts, seemingly through phosphorylation at S1-S3. The differential effects of D6PK and PID on PIN1 polarity had so far been attributed to their differential phosphosite preference for the four PIN1 phosphosites. We have mapped PIN1 phosphorylation at S1-S4 in situ using phosphosite-specific antibodies. We detected phosphorylation at PIN1 phosphosites at the basal (rootward) as well as the apical (shootward) plasma membrane in different root cell types, in embryos, and shoot apical meristems. Thereby, PIN1 phosphorylation at all phosphosites generally followed the predominant PIN1 distribution but was not restricted to specific polar sides of the cells. PIN1 phosphorylation at the basal and apical plasma membrane was differentially sensitive to BFA treatments, suggesting the involvement of different protein kinases or trafficking mechanisms in PIN1 phosphorylation control. We conclude that phosphosite preferences are not sufficient to explain the differential effects of D6PK and PID on PIN1 polarity, and suggest that a more complex model is needed to explain the effects of PID.auxin transport | protein kinase | Arabidopsis | PIN1 | polarity T he phytohormone auxin is a central regulator of plant development and tropic growth (1, 2). Proper plant development strictly requires the directed cell-to-cell transport of auxin, which is achieved by a system of auxin influx and efflux transporters (1). AUXIN RESISTANT1 (AUX1)/LIKE-AUX1 (LAX) proteins are auxin influx transporters and PIN-FORMED (PIN) proteins are auxin efflux transporters that may act together with ABC transporters (3-8). Auxin transport gains its directionality through the often polar distribution of the plasma membrane-resident PIN auxin efflux carriers, PIN1-PIN4 and PIN7 in Arabidopsis thaliana (1, 9). Directional auxin transport results in the formation of cellular auxin maxima and minima that provide essential cues for plant growth and differentiation at the level of individual cells and tissues (1, 2). PIN1 localizes to the basal (rootward) plasma membrane in root stele cells and directs auxin transport toward the root tip (3). PIN2 localizes differentially to the basal (rootward) and apical (shootward) plasma membrane in cortex and epidermis cells, respectively, and the opposing auxin transport streams in cortex and epidermis are important for gravitropic root growth (10, 11).Efficient PIN1-mediated auxin efflux requires activation by phosphorylation (12, 13). In the case...